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PHYTOCHEMICAL INVESTIGATIONS ON
SEA BUCKTHORN (Hippophae rhamnoides) BERRIES
Thesis submitted to
Cochin University of Science and Technology
In partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
IN CHEMISTRY
BY
RANJITH A
AGROPROCESSING AND NATURAL PRODUCTS DIVISION NATIONAL INSTITUTE FOR INTERDISCIPLINARY
SCIENCE AND TECHNOLOGY (CSIR) THIRUVANANTHAPURAM-19, Kerala, INDIA
JUNE 2009
DECLARATION
I hereby declare that the thesis entitled ‘PHYTOCHEMICAL
INVESTIGATIONS ON SEA BUCKTHORN (HIPPOPHAE RHAMNOIDES)
BERRIES’ embodies the results of investigations carried out by me at
Agroprocessing and Natural Products Division of National Institute for
Interdisciplinary Science and Technology (CSIR), Thiruvananthapuram, as
a full-time Research Scholar under the supervision of Dr. C. Arumughan
and that no part of this thesis has been presented before for any other
degree.
Ranjith A
Thiruvananthapuram
June 6, 2009
ACKNOWLEDGEMENT
I am immensely grateful to Dr. C. Arumughan (Guide), Head & Director grade
scientist (Rtd,), Agroprocessing and Natural Products Division, National Institute
for Interdisciplinary Science and Technology (CSIR), Trivandrum for his inspiring
guidance from time to time with utmost patience.
I express my sincere thanks to Dr B. C. Pai, Director, NlIST, Trivandrum for
providing the necessary facilities to carry out this work. I also gratefully
acknowledge the support given by Prof. T. K Chandrasekhar, former Director of
NIIST, Trivandrum. I am also thankful to all present and former members of
Academic Programme Committee of NIIST
I am most grateful to DRDO, Govt of India for conducting the project named
‘CHARAK’ which provided me an opportunity to start my research work as a
Project Fellow in NIIST, Trivandrum.
I am also indebted to CSIR, New Delhi for granting me SRF that enabled me
to complete this investigation.
I would like to express my gratitude to Dr A Sundaresan and Mr. M M
Sreekumar for the support and encouragement extended to my work.
I wish to extend my sincere thanks to Dr Mangala Gowri, Shri. V V
Venugopalan, Dr Padmakumari and Mrs. Prasannakumari for their help in my
work. All the former and present members of Agroprocessing and Natural Products
Division have been extremely helpful and co-operative and I am thankful to one
and all for their kind gesture.
I gratefully acknowledge the help and support received from my friends Mr
Benherlal P S, Mr Sarin Kumar K, Mrs Sreevidya S D, Mr D R Soban Kumar, Dr
Renuka Devi R and Mrs Rajam L.
My heartful thanks to Dr V Vijay Nair, Dr Mangalam S Nair, Dr
Emilia Abraham and Dr U Syamaprasad (NIIST) for their help and support.
I express my sincere thanks to Dr Mohammed Yusuff K K, Dept. of
Applied Chemistry, Cochin University of Science and Technology for his support as
an external expert in Doctoral Committee.
I would like to express my gratitude to all the members of library and
informatics Division, NIIST for their help during the course of the study.
I wish to express my deepest gratitude and appreciation to my Amma, Achan,
Sreeji, Chinju, Aniyettan, Ammoos, Lekshmi and all other family members.
Above all I praise and thank the Almighty for His blessings.
Ranjith A
Dedicated to My Parents, Teachers and Friends
i
CONTENTS
page
LIST OF TABLES iii
LIST OF FIGURES v
CHAPTER 1.0 INTRODUCTION 01
1.1 Preamble 03
1.2 Phytochemicals in Health Care 05
1.3 Phytochemicals as Antioxidants 22
l.4 Sea Buckthorn 46
1.5 Relevance and objectives of the present study 62
CHAPTER 2.0 MATERIALS AND METHODS 67
2.1 Sea Buckthorn Berries 69
2.2 Chemicals and Reagents 69
2.3 Proximate Composition of Berries 70
2.4 Extraction of Lipids-Christie’s Method 71
2.5 GC Profiling of Fatty Acids 72
2.6 Total Carotenoids 72
2.7 HPLC Profiling of Tocopherols/Tocotrienols 72
2.8 HPLC Profiling of Sterols 73
2.9 HPLC Quantification of Vitamin C 73
2.10 Organic Acid Analysis 74
2.11 Total Phenolics 75
2.12 Flavonoids 75
2.13 Standardization of HPLC-DAD Analytical Method
for Phenolic Acids
79
2.14 Analysis of Proanthocyanidins 81
2.15 Processing of Fresh Sea Buckthorn Berries 84
2.16 Super Critical Carbondioxide Extraction of Seed
oil
86
2.17 Activity Guided Fractionation of Sea Buckthorn 86
ii
Berries
2.18 In vitro Antioxidant Capacity Assays 89
CHAPTER 3.0 RESULTS AND DISCUSSION 93
3.1 Phytochemical Profiling of H. rhamnoides Berries
of Trans-Himalayan Origin
95
3.2 Phytochemical Profiling of Berries of Major Sea
Buckthorn Species in Indian Trans-Himalayas
135
3.3 Processing of Fresh Sea buckthorn Berries 147
3.4 In Vitro Antioxidant Capacity Evaluation of H.
rhamnoides Berries
163
CHAPTER 4.0 SUMMARY AND CONCLUSION 203
REFERENCES 211
ABBREVIATIONS 219
PUBLICATIONS 221
iii
LIST OF TABLES
Table
No.
Caption page
1.1 Classification of phenolic compounds based on structure 09
1.2 Chemical sources of ROS and RNS 26
1.3 Biological sources of ROS and RNS 27
1.4 Role of free radical reactions in biology 29
1.5 Role of free radical reactions in diseases and ageing 29
3.1 Proximate composition of H. rhamnoides berry pulp and seed 98
3.2 FA composition of pulp and seed oils of H. rhamnoides 99
3.3 Carotenoid and tocol content of pulp and seed oils of H. rhamnoides berries
102
3.4 Sterols in H rhamnoides pulp and seed oils 107
3.5 Vitamin C and organic acids content in H. rhamnoides fresh berries
111
3.6 Total polyphenols, flavonols and PAs in H. rhamnoides berries 114
3.7 Flavonoids in H. rhamnoides berries 116
3.8 Yield, total polyphenols and PAs in crude and purified PA extracts and ADP of H. rhamnoides in SB berries
123
3.9 Detection wave length, TR and linear regression data of phenolic acids
129
3.10 Precision, accuracy and recovery of phenolic acids. 130
3.11 Free and bound phenolic acids in berry pulp, kernel and seed coat 131
3.12 Seed content and proximate composition of SB berries of H. rhamnoides, H. salcifolia and H. tibetana species from India
136
3.13 Vitamin. C, polyphenol and flavonol content of SB berries of H.rhamnoides, H. salcifolia and H. tibetana Species from India.
139
3.14 FA composition of pulp oils of SB berries from H. rhamnoides, H. salcifolia and H. tibetana Species from India
141
3.15 Carotenoids, tocopherols and tocotrienols composition of pulp oils of SB berries of H. rhamnoides, H. salcifolia and H. tibetana species from India
145
3.16 Results of process development trials of fresh sea buckthorn berries.
149
3.17 Compositional analysis of process streams 150
3.18 Polyphenols and flavonoids in the pulp of berries and juice 152
iv
3.19 pH, titrable acidity, organic acids and vitamin C in the pulp of berries and juice
154
3.20 FA composition of pulp oil 156
3.21 Carotenoid, tocopherol, tocotrienol and sterol content in the pulp of berries and pulp oil
156
3.22 FA composition of hexane and supercritical CO2 (SC- CO2) extracted seed oils
159
3.23 Yield, carotenoid, tocopherol, tocotrienol and sterol content in the hexane and SC- CO2 extracted seed oils
159
3.24 Extraction efficiency in terms of yield of extract of different solvents on sequential extraction.
164
3.25 TPC and in vitro antioxidant capacities of SB berry extracts and reference compounds
171
3.26 Yield, phenolic content and in vitro AO capacities of KME fractions and reference compounds
177
3.27 HPLC-ESI-APCI profile of flavonoid glycoside in KMEF 181
3.28 In-vitro AO capacities of flavonols 187
3.29 Phenolic acid composition of KMWF. 189
3.30 Concentration of reaction products after acid catalyzed cleavage of KMWF proanthocyanidins in presence of phloroglucinol.
193
3.31 Yield and composition of KMWF fractions. 197
3.32 In-vitro AO capacities of KMWF fractions 201
v
LIST OF FIGURES
Figure
No.
Caption page
1.1 Schematic illustration of the origin of various phenolics from simple phenyl propanoids
17
1.2 Interrelationships among flavonoid monotypes 18
1.3 Structures of some biologically active phytochemicals 20
1.4 Structures of tannins and hexahydroxydiphenyl unit 21
1.5 Molecular orbital diagram of different oxygen derived species
25
1.6 Structures of some stable free radicals and synthetic AO compounds
34
1.7 Structures of L-ascorbic acid and dehydroascorbic acid 37
1.8 Structures of taurine and hypotaurine 39
1.9 Lipoic acid 40
1.10 Sea buckthorn 46
2.1 Processing of fresh SB berries 85
2.2 Activity guided extraction scheme of SB berries 88
3.1 Anatomy of sea buckthorn berries 96
3.2 Tocopherols and tocotrienols 105
3.3 HPLC profile of tocols in H. rhamnoides pulp oil 106
3.4 HPLC profile of sterols in H. rhamnoides seed oil 108
3.5 Structures of major sterols in SB berries 108
3.6 Structure of major organic acids in SB berries 112
3.7 HPLC chromatogram of organic acids in H. rhamnoides berry pulp
112
3.8 Structures of major flavonols in SB berries 116
3.9 HPLC-DAD chromatogram of flavonols in H. rhamnoides berry pulp at 270 nm
117
3.10 Effect of different solvents on the extraction of PAs from SB berry pulp, kernel and seed coat.
120,121
3.11 Phenolic acids 124
3.12 HPLC-DAD chromatograms of phenolic acids at different wave lengths
132
3.13 HPLC-DAD chromatograms of phenolic acids in H. rhamnoides kernel extract at different wave lengths
133
vi
3.14 ABTS 167
3.15 DPPH radical scavenging efficacies of H. rhamnoides berry extracts and reference compounds.
172
3.16 Fe(III) reduction capacities of H. rhamnoides berry extracts and reference compounds.
173
3.17 Structures of quercetin, kaempherol and isorhamnetin and their glycosides in SB seeds
182
3.18 HPLC-MS/MS total ion chromatogram of flavonoid glycosides in KMEF
183
3.19 Structures of reaction products obtained by the acid catalyzed cleavage of SB PAs in presence of phloroglucinol.
194
3.20 Total ion chromatogram of depolymerisation reaction mixture of purified PAs from KMWF
195
1
Chapter 1.0
INTRODUCTION
2
The highest education is that which does not
merely give us information
but makes our life in harmony with all existence
....Tagore
Introduction
3
1.1. Preamble
Overwhelming epidemiological evidences indicate that plant based
diets afford protection against the development of certain chronic diseases,
particularly cancer. Plants provide rich source of phytochemicals and
phytopharmaceuticals and have been used since antiquity to treat and manage
different diseases. The active plant constituents developed as a part of their
own defense mechanism seems to contribute to man’s health. Phytochemicals
are secondary metabolites in plants and many of them are incorporated into
foods or used as food supplements or nutraceuticals or as pharmaceuticals that
can function in vivo to complement or boost the endogenous defense system.
Thus functional foods, nutraceuticals or phytoceuticals capable of providing
additional physiological benefits such as preventing or delaying the onset of
chronic diseases are now as considered alternative health care.
The mounting cost of modern health care and contra indications of
chemical entities based therapies coupled with recent experimental and
epidemiological evidences in favor of phytochemicals have encouraged
people to accept the concept of alternative health care. The world trade in
herbal products are growing at a faster pace as a result of popularization of
complimentary and alternative medicinal systems, but the product’s true
information suffer from lack of active principles, validation etc. Since the
plants and plant products are subjected to wide variation in their
phytochemical profile due to variety, geo climatic conditions, maturity, post
harvest processing, storage, stability etc, it is extremely important to conduct
detailed investigations on the composition and physiological significance of
medicinal plants and standardize the formulations based on ingredients.
Introduction
4
Although natural products have been a well spring of drugs and drug
leads for decades, synthetic drugs have dominated in modern western
medicine for the past two decades due to the emergence of promising and
exciting advancements and technologies associated with combinatorial
chemistry. The diminished productivity of de novo molecules towards drugs
or drug leads and recent developments in the separation and analytical
techniques reclaims the attention of researchers on natural product drug
discovery. According to a recent survey 61 % of small-molecule new
chemical entities introduced as drugs world wide during 1981-2002 can be
traced to or were inspired from natural products. In case of antibacterials and
anticancer agents the productivity was higher than 70%. Despite the
spectacular results obtained during the past years in the development of new
drugs through biotechnology, genetic engineering and bioinformatics, natural
products still have a very important role to play in drug discovery. In fact,
only about 10% of the existing 350,000 plant species have been investigated
from a phytochemical and pharmacological point of view. Nature’s
architecture (not only plants but also marine organisms, insects and even
animals) provides an unpredictable range of skeletal types and novel
substances that it is of immense value to evaluate as many natural products as
possible in order to find sources of new drugs, new lead compounds, new
pesticides and new compounds for animal health.
Introduction
5
1.2. Phytochemicals in Health Care
All living organisms are required to transform and interconvert a vast
number of organic compounds to enable them to live, grow, reproduce and
survive. They need to provide themselves with energy and supply precursors
to construct their own tissues. An integrated network of enzyme mediated and
carefully regulated reactions are involved for this purpose. Collectively they
are referred as metabolism and the reaction steps involved are termed
metabolic pathways. Despite the extremely varied characteristics of living
organisms, the pathways for the modification and synthesize of
carbohydrates, proteins, fats, and nucleic acids are in general essentially the
same in all organisms collectively described as primary metabolism, the
compounds involved in the pathways are termed primary metabolites.
Glycolysis, oxidation of fatty acids (β-oxidation), digestion of proteins etc
thus, are classified as primary metabolism. The metabolism concerned with
the compounds which have a limited distribution in nature and is specific to
certain organism is referred to as secondary metabolism and such compounds
are called secondary metabolites. Secondary metabolites are generally
produced in response to climatic stress, infection, defense against predators,
for propagation etc. Most of the pharmacologically active natural products
such as phenolic compounds, alkaloids, terpenoids etc are products of
secondary metabolism. The phytochemicals those generally known as
secondary metabolites of pharmacological significance are discussed below.
1.2.1. Lipids and their derivatives
Water insoluble biomolecules but soluble in non-polar solvents are
broadly termed as lipids. There are some limitations in this classification as
most organic compounds fall in this category and many classical fatty acids
Introduction
6
have significant solubility in water. A more constricted classification of lipids
is to simply classify them as fatty acids and their derivatives and to treat other
hydrocarbons based natural products separately
1.2.1.1. Hydrocarbons: Hydrocarbons comprise a relatively small group of
natural products with least polar nature. Aliphatic hydrocarbons in plants
usually have odd number of carbon atoms and are mainly derived through the
decarboxylation of fatty acids (1). Aliphatic and aromatic as well as saturated
and unsaturated hydrocarbons are seen in plants. The highly branched
hydrocarbons derived from isoprene are considered as a separate group
(terpenoids). Saturated hydrocarbons are widely distributed as the waxy
coating on leaves and surface of fruits. As the chain length and unsaturation
increase hydrocarbon become more waxy and solid in room temperature. The
simplest unsaturated hydrocarbon ethylene is an important plant hormone
inducing abscission and ripening of fruits. Larger unsaturated hydrocarbons
are also common in plants. Polyacetylenes are unique group of plant
hydrocarbons with one or more acetylene linkages. Polyacetylenes are likely
to be derived through the enzymatic dehydrogenation of corresponding
olefins. Polyacetylenes with 14 to 18 carbon atoms are found in higher plants.
Most of the polyacetylenes are toxic in nature and are mainly involved in
plants defense mechanism. Cicutoxin in Oceanthe crocata (2) and falcarinol
(Fig. 1.3) in domestic carrot (Daucos carota) root (3) are examples.
1.2.1.2. Alcohols: Large varieties of volatile aliphatic alcohols occur in small
quantities in plants as a part of essential oils. All of the straight chain alcohols
from C1 to C10 are found in plants either in free or esterified forms. Several
larger alcohols such as ceryl alcohol are regular constituents of cuticular wax.
Aliphatic alcohols containing cis-3-hexene-1-ol moiety have characteristic
odors and are of interest to the fragrance industry (4).
Introduction
7
1.2.1.3. Aldehydes and ketones: Low and medium molecular weight
aldehydes and ketones occur as a part of volatile oils in plants. Citrus plants
and bergamot (Monarda didyma) on cold pressing yield essential oils rich in
aldehydes and ketones. Citral, nootketone, octanal etc are examples of
industrially important carbonyl compounds (5).
1.2.1.4. Fatty acids (FAs): Natural FAs may contain from 4 to 30, or even
more, carbon atoms, the most abundant being those with 16 or 18 carbons in
plants. FAs are mainly found as esters with glycerol and are called fats or oils,
depending on whether they are solid or liquid at room temperature. Most
natural fats and oils are composed largely of mixed triglycerides. Animal fats
contain a high proportion of glycerides of saturated FAs and tend to be solids,
whilst those from plants and marine organisms contain predominantly
unsaturated FA esters and tend to be liquids. Selective cis isomerisation of
FAs in plants diminishes the close association of molecules and facilitates to
maintain fluidity of cellular membranes.
1.2.1.5. Terpenoids: The terpenoids form a large and structurally diverse
family of natural products derived from C5 isoprene units, joined in a head to
tail fashion therefore these are also referred to as ‘isoprenoids’. Typical
structures contain carbon skeletons represented by (C5)n, and are classified as
hemiterpenes (C5), monoterpenes (C10, eg; geraniol) (Fig-1.3), sesquiterpenes
(C15, eg; farnesol), diterpenes (C20, eg; geranylgeraniol), sesterterpenes (C25),
triterpenes (C30, eg; Squalene) and tetraterpenes (C40, eg; phytene). Higher
polymers are encountered in materials such as rubber. Relatively few of the
natural terpenoids exactly encounter the simple concept of a linear head-to-
tail combination of isoprene units. Most terpenoids are modified further by
cyclization reactions e.g. menthol, bisabolene and taxadiene. The linear
arrangement of isoprene units can be more difficult to appreciate in many
other structures like sterols when rearrangement reactions have taken place.
Introduction
8
1.2.1.6. Steroids: The steroids are modified triterpenoids containing the
tetracyclic ring system of lanosterol,. Cholesterol (Fig. 1.3) exemplifies the
fundamental structure of steroids. Modifications on the side-chain result in a
wide range of biologically important natural products. Steroids include a
variety of bioactive compounds such as sterols, steroidal saponins, cardio
active glycosides, bile acids, corticosteroids and mammalian sex hormones.
Many natural steroids together with a considerable number of synthetic and
semi-synthetic steroidal compounds are routinely employed in medicine (6,7)
1.2.1.7. Carotenoids: Carotenoids are the most common tetraterpenoids with
wide distribution in plant kingdom. Carotenoids are generally derived from
lycopene through the cyclization of end groups. β-carotene (Fig-1.3) is the
most common carotenoid in higher plants. Carotenoids not only impart bright
colors to plants, but also involved in photosynthesis and antioxidant (AO)
defense mechanism.
1.2.2. Aromatics
Compounds with aromatic rings contribute a major share of natural products.
Aromatic compounds are involved in a wide spectrum of physiological
functions of plants such as color, photosynthesis, microbial deterrence and
structural composition. Aromatic compounds are formed through several
biosynthetic routes including polyketide and shikimate pathways as well as
from terpenoids. The vast majority of aromatic compounds are phenols.
1.2.2.1. Phenolic Compounds
They are compounds that have one or more hydroxyl groups attached
directly to an aromatic ring. Because of the aromatic ring, the hydrogen of the
phenolic hydroxyl is labile, which makes phenols weakly acidic. Polyphenols
are compounds that have more than one phenolic hydroxyl group attached to
Introduction
9
one or more benzene rings. Phenolic compounds are characteristic of plants
and as a group they are usually found as esters or glycosides rather than as
free compounds. The term ‘phenolics’ covers a large and diverse group of
chemical compounds. These compounds can be classified in a number of
ways. Harborne and Simmonds classified these compounds into groups based
on the number of carbons in the molecule (Table 1.1) (8). Origins of various
phenolics from simple phenyl propanoids are illustrated in Figure 1.1.
Table 1.1. Classification of phenolic compounds based on structure.
Structure Class
C6 Simple phenolics
C6-C1 Phenolic acids and aldehydes
C6-C2 Acetophenones and phenyl acetic acids
C6-C3 Cinnamic acids, cinnamyl alcohols and cinnamyl aldehydes
C6-C1-C6 Benzophenones
C6-C2-C6 Stilbenes
C6-C3-C6 Flavonoids
C18 Betacyanins
C30 Biflavonoids
C6, C10 & C14 Quinines
Dimmers or oligomers Lignans and neolignans
Oligomers or polymers Tannins
Polymers Phlobaphens
(i) Simple phenolics: Simple phenolics are substituted phenols. Catechol
(1,2-dihydroxybenzene), resorcinol (1,3-dihydroxybenzene) and
phloroglucinol (1,3,5-trihydroxybenzene) etc are typical examples for simple
phenolics.
Introduction
10
(ii) Phenolic acids: Phenolic acids are important phenolic compounds of non
flavonoid family, which constitute a large group of phenolic compounds in
plants. Phenolic acids includes two main groups namely, hydroxybenzoic
acid and hydroxycinnamic acid derivatives. They differ according to the
number and position of hydroxylation and methoxylation in aromatic ring
(Figure-1.3). Phenolic acids are distributed as their free and bound forms in
nature, more often bound forms occur as their esters and glycosides. Phenolic
acids are reported to have a wide spectrum of pharmacological activities
including AO, antimutagenic, antitumor and anticarcinogenic properties
(9,10).
(iii) Coumarins: Coumarins have a C6-C3 skeleton, with an oxygen
heterocycle as part of the C3-unit. There are numerous coumarins, many of
which play a role in disease and pest resistance, as well as UV-tolerance.
Umbelliferone (Figure 1.3) is a popular coumarin used in enzyme assays.
Isocoumarins, such as bergenin have a structure similar to coumarins, but the
position of the oxygen and carbonyl groups within the oxygen heterocycle are
reversed. Isocoumarins also play a role in defense responses (11).
(iv) Flavonoids : Flavonoids are C15 compounds all of which have the
structure C6-C3-C6. Flavonoids may be grouped into different classes based
on their general structure (Figure 1.3).
Chalcones and dihydrochalocones have a linear C3-chain connecting
the two rings. The C3-chain of chalcones contains a double bond, whereas the
C3-chain of dihydrochalcones is saturated. Chalcones, such as butein, are
yellow pigments in flowers. An example of a dihydrochalcone is phloridzin
(phloretin-2′-O-D-glucoside), a compound found in apple leaves, and which
has been reported to have anti-tumor activity (12). Aurones are formed by
cyclization of chalcones, whereby the meta-hydroxyl group reacts with the α-
Introduction
11
carbon to form a five-member heterocycle. Aurones are also yellow pigments
present in flowers.
Typical flavonoids, such as flavanone, have a six-membered
heterocycle. The A-ring originates from the condensation of three malonyl-
CoA molecules, and the B-ring originates from p-coumaroyl-CoA. These
origins explain why the A-ring of most flavonoids is either m-dihydroxylated
or m-trihydroxylated. In typical flavonoids one of the m-hydroxyl groups of
the A-ring contributes the oxygen to the six member heterocycle. The oxygen
heterocycle of typical flavonoids may be a pyran, pyrylium, or pyrone ring.
The B-ring is typically monohydroxylated, o-dihydroxylated, or vic-
trihydroxylated. The B-ring may also have methyl ethers as substituents.
The heterocycle of flavanones also contains a ketone group with
saturated C2-C3 carbon-carbon bond (eg; naringenin). Dihydroflavonols are
known as flavanonols and often occur in association with tannins in
heartwood (eg: Taxifolin or dihydroquercetin).
Flavan-3,4-cis-diols are referred to as leucoanthocyanidins. They are
synthesized from flavanonols via a reduction of the ketone moiety on C4.
Examples are leucocyanidin and leucodelphinidin. These compounds are
often present in wood and play a role in the formation of condensed tannins.
Because of their completely saturated heterocycle, leucoanthocyanidins,
together with flavan-3-ols are referred to as flavans. Catechin and
gallocatechin are examples of flavan-3-ol. The ‘gallo’ in the latter compound
refers to the vic-tri-hydroxy substitution pattern on the B-ring. Unlike most
other flavonoids, the flavans are present as free aglycones or as polymers of
aglycones. Catechins can also be found as gallic acid esters that are esterified
at the 3′ hydroxyl group.
Introduction
12
The heterocycle of flavones contains a ketone group, and has an
unsaturated carbon-carbon bond. Flavones are common in angiosperms. The
most widely distributed flavones in nature are kaempherol
(5,7,4′hydroxyflavone2), quercetin (5,7,3′,4′ hydroxyflavone), and myricetin
(5,7,3′,4′,5′hydroxyflavone).
Flavonoids with pyrilium cation are refered to as anthocyanidins.
Widely distributed, colored anthocyanins such as pelargonidin, cyanidin,
delphinidin, petunidin, and malvidin are found as their aglycones. The most
common anthocyanidin is cyanidin. These compounds are present in the
vacuoles of colored plant tissues such as leaves or flower petals. The color of
the pigment depends on the pH, metal ions present, and the combination of
substituted sugars and acylesters. Different colors can also result from the
presence of combinations of several anthocyanidins.
Isoflavonoids possess a rearranged flavonoid skeleton. Variety of
structural modifications of the skeleton lead to different groups including
isoflavones, isoflavonones and rotenone. The isoflavones are common in
legume family Fabaceau. Genestein from soybean and coumstrol from
Medicago sativa are phytoestrogens (13).
(v) Biflavonyls: Biflavonyls have a C30 skeleton. They are dimers of
flavones such as apigenin or methylated derivatives and are found in
gymnosperms. The most familiar is ginkgetin from Ginkgo biloba.
(vi) Benzophenones and xanthones: Benzophenones and xanthones have a
C6-C1-C6 structure. Xanthones are yellow pigments in flowers.
Introduction
13
(vii) Stilbenes: Stilbenes have a C6-C2-C6 structure and are localized in
woody stems and seeds. Resveratrol, a well known stilbene found in grape is
reported to have cardioprotecive effects.
(viii) Benzoquinones, anthraquinones and naphthaquinones:
Benzoquinones, such as 2,6-dimethoxybenzoquinone are present in root
exudates of maize and stimulate parasitic plants to form haustoria (14).
Ubiquinones, the benzoquinone with isoprenoid sidechains, are also known as
Coenzyme Q and have a role in electron transport in the mitochondria.
Naphthaquinones are rare. Among the naphthaquinones juglone is relatively
common and it is found in walnuts. Anthraquinone is the most widely
distributed of the quinones in higher plants and fungi. The anthtraquinone
emodin occurs as a rhamnoside in rhubarb roots.
(ix) Betacyanins: Betacyanins are red pigments and account for the red color
of beets (Beta vulgaris). They are unique compounds to the Centrospermae
with absorption spectra that resembles anthocyanins, but they contain nitrogen
eg; betanidin. Betacyanins are normally found as their glycosides.
(x) Lignans: Lignans are dimers or oligomers that result from the coupling of
monolignols – p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol,
with coniferyl alcohol being the most common monolignol used in lignan
biosynthesis. Lignans are present in ferns, gymnosperms and angiosperms.
They are localized in woody stems and in seeds and play a major role in
insect deterrence. The term lignan typically refers to dimers of monolignols
that are linked via an 8-8′ (β-β′) bond, whereas the term neolignan refers to
dimers and oligomers that contain bonds other than the 8-8′ bond. Most
lignans are optically active, and typically only one enantiomer is found in a
given species. Examples of lignans include (+)-pinoresinol, (+)-sesamin, and
(–)-plicatic acid.
Introduction
14
(xi) Lignin: Lignin, the second most abundant bio-polymer on earth (after
cellulose) is a phenolic polymer. It provides structural support to plants,
facilitates water transport through the vascular tissue and acts as a physical
barrier against insects and fungi. Lignin is synthesized primarily from three
monolignol precursors: p-coumaryl alcohol, coniferyl alcohol and sinapyl
alcohol. Comparatively small amounts of additional compounds such as
coniferaldehyde, sinapaldehyde, dihydroconiferyl alcohol, 5-hydroxyconiferyl
alcohol, tyramine ferulate, p-hydroxy-3-methoxybenzaldehyde, p-
hydroxybenzoate, p-coumarate and acetate are also found in lignin (15)
(xii) Tannins: Tannins comprise a group of compounds with a wide diversity
in structure and commonality in their ability to bind and precipitate proteins.
The process of tanning animal skin to make leather utilize tannin and hence
the name tanning. In Indian, Japanese and Chinese natural medicine tannins
have been used as anti-inflammatory and antiseptic compounds. In wine and
beer production, tannins are used to precipitate proteins. Tannins are abundant
in many different plant species particularly in oak (Quercus spp.), chestnut
(Castanea spp.), staghorn sumac (Rhus typhina), and fringe cups (Tellima
grandiflora). Tannins are also present in leaves, bark, and fruits, and are
known to protect the plant against infection and herbivory. Tannins can be
classified under three groups: condensed tannins, hydrolysable tannins, and
complex tannins. Hydrolysable tannins are further divided in to gallotannins
(GT) and elligitannins (ET).
(a) Gallotannins (GTs): GTs (Figure 1.4) are hydrolysable tannins with a
polyol core (a compound with multiple hydroxyl groups) substituted with 2-
12 gallic acid residues. GT contains the characteristic meta-depside bonds
between gallic acid residues. This bond is more labile than an aliphatic ester
bond, and can be methanolyzed with a weak acid in methanol. The most
Introduction
15
commonly found polyol is D-glucose, although some GT contain catechin and
triterpenoid units as the core polyol.
(b)Condensed tannins: Condensed tannins are also referred to as
proanthocyanidins (PAs). PA are mixtures of oligomers and polymers of
flavan-3-ols, mainly formed through C4 C8 and/or C4 C6 (B type)
or linked through an ether bond between C2 C7 (A type). On hydrolysis
under harsh conditions, such as heating in acid, PA yields anthocyanidins. An
example of PA is procyanidin B2 (epicatechin-(4β→8′)-epicatechin).
Polymers are formed through the action of acids or enzymes. Polymers made
up of more than 50 catechin units have been identified. The protein
precipitation capacity of PA has importance in wine making, where a high
level of condensed tannins, especially in red wines, can result in the dry
feeling in mouth.
(c) Ellagitannins (ETs): ETs are also hydrolysable tannins derived from
pentagalloylglucose, but unlike GT, they contain additional C-C bonds
between adjacent galloyl moieties in the pentagalloylglucose molecule. This
C-C linkage is formed through oxidative coupling between the two adjacent
galloyl residues, and results in the formation of a hexahydroxydiphenoyl
(HHDP) unit. The name ET is derived from ellagic acid, which is formed
spontaneously from hexahydroxydiphenic acid in aqueous solution via an
intra-molecular esterification reaction.
(d) Complex tannins. Complex tannins are defined as tannins in which a
catechin unit is bound glycosidically to either a GT or an ET unit. As the
name implies, the structure of these compounds can be very complex (eg;
acutissimin A (Figure 1.4)). Acutissimin A is a flavogallonyl unit bound
glucosidically to C-1, with an additional three hydrolyzable ester bonds to a
D-glucose-derived open-chain polyol. This complex tannin is formed during
Introduction
16
the aging process of red wine, whereby the catechin unit originates from the
grapes, and the ET originates from the oak barrels. Acutissimin A has been
shown to be a powerful inhibitor of DNA topoisomerase II, an enzyme
required for the division of cancer cells, and a target for chemotherapeutic
drugs (16).
(e) Phlobaphenes: Phlobaphenes are phenolic polymers present in floral
organs of maize (Zea mays L.), Accumulation of phlobaphenes results in red
pigmentation. Certain lines of sorghum (Sorghum bicolor L. (Moench)) also
produce phlobaphenes. The structure of phlobaphenes is poorly understood.
These compounds are believed to be polymers of flavan-4-ols, notably
apiferol and luteoferol. The polymerization is thought to be under chemical,
rather than enzymatic control, and give rise to a polymer in which the
monomers are linked via a 4-8′ linkage. The C-C bonds between the flavan-
4-ol monomers are difficult to break, which makes the structural elucidation
of phlobaphenes difficult.
Introduction
17
Figure 1.1 Schematic illustration of the origin of various phenolics from simple phenyl propanoids
Introduction
18
Figure 1.2 Interrelationships among flavonoid monotypes
Introduction
19
1.2.3. Amines and alkaloids
1.2.3.1 Amines: Compounds containing nitrogen as a part of their side chain
are generally referred to as amines. These molecules impart fishy odor and
believed to act as insect attractants. Aliphatic polyamines such as putriscine
(NH2(CH2)4NH2), agmatin (NH2(CH2)4NHC(=NH)NH2), and spermidine
(NH2(CH2)3NH(CH2)4NH2) are also found in plant kingdom. These
polyamines are thought to have many biological functions in plant including
acting as plant hormones and are invariably found complexed with nucleic
acids (17). Most of the plant aromatic amines are physiologically active.
Mescalin (from Lophophora willimasii) is a potent hallucinogen.
Noradrenaline, histamine and serotonin occurring in common plants are
critical to brain metabolism (18).
1.2.3.2. Alkaloids: Alkaloids are classically defined as the plant derived basic
compounds with pharmacological effects and contain one or more nitrogen
atoms. In practice most of the nitrogen containing secondary metabolites are
considered as alkaloids unless they may be readily classified otherwise.
Alkaloids impart a wide spectrum of physiological effects in plants and
animals. Alkaloids in plants serve as chemo protective, anti-herbivory agents
or as growth regulator such as indole-3-acetic acid. Reserpine (from
Rauwolfia serpentina) is an antihypertensive alkaloid. Cocaine is a local
anesthetic and a potent central nervous system stimulant. Caffeine is one of
the world’s most popular additive drugs. Quinine derived from cinchona trees
have been used as an anti-malarial drug. Morphine, the principal alkaloid of
opium poppy (Papver somniferum) is a potential narcotic analgesic.
Strychnine from Strychnos nux vomica is a strong poison. Colchinine from
(Colchicum autmuale) has been used to treat gout for 2000 years. Atropine as
a smooth muscle relaxant is used to dilate the pupil before eye examination
and is also used for the treatment of ambylopia (lazy eye). Camptothecine, a
quinoline alkaloid from Chinese tree of joy (Camptotheca accuminata) is well
known for its antiapoptic activity. Papaverine is used as a vasodialator (19).
Introduction
20
HO
Falcarinol
OH
Geraniol
β-carotene
HO
H
HH
Cholesterol
CO2H
HO
HO
HO
Gallic acid
HO
HO
CO2H
Caffiec acid
O OHO
Umbelliferone
Squalene
O
O
OH
OH
OH
OH
HO
Quercetin
HN
N
O
O
O
O
O
O
O
O
H
H
H
O
reserpine
N
N
OO
OH
O
camptothecin
Figure 1.3: Structures of some biologically active phytochemicals
O
O
O
O
HO
HO
OH
OH
Ellagic acid
Introduction
21
Gallotannin
Condensed tannin
(proanthocyanidin)
Hexahydroxydiphenoyl (HHDP) unit
Complex tannin Phlobaphenes
Figure 1.4 Structures of tannins and hexahydroxydiphenyl unit
Introduction
22
1.3. Phytochemicals as antioxidants
1.3.1 Free Radicals
Free radicals can be defined as molecules or molecular fragments
containing one or more unpaired electrons. This unpaired electron(s) usually
seeks other electron(s) to become paired and thus, the free radicals in general
are highly reactive and less stable. Their life times are extremely short in
solutions, but they can be kept frozen for relatively long periods within the
crystal lattice of other molecules. The lifetime of a radical depends not only
on its inherent stability, but also on the conditions under which it is generated.
A stable radical is inherently stable; a persistent radical has a relatively long
lifetime under the conditions at which it is generated, although it may not be
very stable.
Radicals can be characterized by several techniques, such as spin
resonance spectroscopy, mass spectrometry (20), and Step-Scan Time-
Resolved Infrared Spectroscopy (21). Electron Spin Resonance (ESR) also
called Electron Paramagnetic Resonance (EPR) spectroscopy is an important
general technique used for the characterization of free radicals. ESR
spectroscopy utilizes the resultant magnetic moment of the unpaired electrons
and their behavior in a strong magnetic field. Since only the free radicals
respond to ESR, the method can be used to detect and quantify the free
radicals. Another important magnetic spectroscopy NMR can also be used to
detect the presence of free radicals during the course of reactions, through
examining the variations in NMR signals due to chemically induced dynamic
molecular polarization (CIDNP) (22).
The stability of free radicals depends on the field strengths,
hyperconjugation, resonation possibilities and steric factors. Generally the
Introduction
23
order of the stability of simple free radicals are primary <secondary <tertiary
<allyl <benzyl. Triphenyl radical is stable enough to exist in solution. Steric
hindrance to dimerisation is thought to be the major reason for its stability.
Diphenyl picryl hydrazyl (DPPH) (Figure 1.6) is a stable free radical that can
be kept for years and used as a probe for radical scavenging assays. TEMPO
(2,2,6,6-tetramethylpiperidine-1-oxyl free radical) (Figure 1.6) is a stable
nitrosyl radical used in chemical reactions and for spin trapping.
A number of biradical (or diradicals) are also known. Biradicals are
short lived species with life time less than 1s. Biradical of 3,5-di-tert-butyl-
3’(N-tert-N-aminoxy)-4-oxybiphenyl is reported to be stable for weeks.
Radicals with both electrons on same carbon are known as carbenes. Carbenes
are highly recative species with very short life time (< 1s). Methylene and
dichloromethylene are examples for carbenes which are extensively used in
organic chemistry.
Many metabolic reactions in biological systems involve oxygen, and
most of the oxygen is reduced to water. However, if the reduction is
incomplete a series of reactive free radicals are formed. Free radicals thus
formed may trigger free radical chain reactions. Environmental factors such as
ionizing radiations, food, cigarette smoke, trace metals etc also contribute to
the free radical generations. The common free radicals in our body include
reactive oxygen species (ROS), reactive nitrogen species (RNS) and some
sulphur- centered radicals
1.3.1.1 Reactive Oxygen Species (ROS)
Reactive oxygen species (ROS) ie; radicals derived from oxygen,
represent the most important class of radical species generated in living
system. Oxygen itself is a diradical with 2 unpaired electrons residing in
antibonding ╥ orbitals. This distribution of electrons makes it impossible for
Introduction
24
oxygen to accept a spin-matched pair of electrons. However one of its
unpaired electrons may undergo a spontaneous spin-reversal to make the
pairing possible. At ordinary collision frequency, the period of contact is too
short to spin reversal to occur, imposing a kinetic barrier to oxidative reaction.
This kinetic barrier saves us from reacting explosively with an atmosphere of
huge thermodynamic oxidizing potential.
Occasionally, under normal biological conditions, oxygen does manage
to capture electrons from other molecules resulting in the formation of free
radicals. The one electron reduction product of oxygen is the superoxide
anion radical (O2●-) (Figure 1.5). It is formed in many autoxidation reactions
and by the electron transport chain and is considered as a primary ROS. It is
less reactive and less harmful in physiological systems but can react with
other molecules to generate secondary ROS either directly or through
enzymatic or non-enzymatic process. It undergoes dismutation to form
hydrogen peroxide (Figure 1.5) spontaneously or by enzymatic catalysis. It
can release Fe2+ from iron-sulphur proteins and ferritin (23). If two electrons
are transferred, the product is hydrogen peroxide (H2O2). It is mainly formed
by dismutation of O2- or by direct reduction of O2. It is lipid soluble and thus
able to diffuse across membranes. Even though H2O2 is a stable non-radical, it
act as precursor for the most reactive ROS namely hydroxyl radical (OH●).
Hydroxyl radical is the three electron reduction state of oxygen. It is one of
the most potent oxidant known and has a very short half-life of approximately
10-9 S. Fenton’s reactions and decomposition of peroxynitrite result hydroxyl
radicals. It is extremely reactive and attacks most of the cellular components.
Alkoxy (RO●) and peroxy (ROO●) radicals and organic hydro peroxide
(ROOH) are formed in association with lipid oxidation reactions.
Hypochlorous acid (HOCl) is formed from hydrogen peroxide by
myeloperoxidase. It is lipid soluble and highly reactive. It readily oxidize
protein constituents, including thiol groups, amino groups and methionine.
Introduction
25
Peroxynitrate (OONO-) formed in a rapid reaction between O2- and NO- is
also considered as active oxygen species. Protonation of peroxynitrate forms
peroxynitrous acid, which can undergo hemolytic cleavage to form OH● and
NO2. Chemical formation and exogenous sources of various ROS are
summarized in Table 1.2
Figure 1.5. Molecular orbital diagram of different oxygen derived species.
Σ*2P
╥*2P
╥2P
Σ2p
Σ*2S
Σ2S
Σ*1S
Σ1S
Ground State O2
Singlet O2 1∆g O2
Superoxide ion
Peroxide ion
Singlet O2 1ΣgO2
1.3.1.2. Reactive Nitrogen Species (RNS)
Nitric oxide (NO●), is a small molecule that contains one unpaired
electron and is therefore a radical. Nitric oxide is generated in biological
tissues by specific nitric oxide synthases (NOSs) which metabolize arginine to
citrulline with the formation of NO● via five electron oxidative reaction. It is a
highly reactive molecule with a half-life of only a few seconds in an aqueous
environment. It has a greater stability in an environment with a lower oxygen
Introduction
26
concentration. As it is soluble in aqueous and lipid media, it readily diffuses
through the cytoplasm and plasma membrane.
Table 1.2. Chemical sources of ROS and RNS (24-26)
ROS/RNS Chemical formation/exogenous sources
Singlet oxygen Photosensitized oxidation. The reactions like
OCl▬ + H2O2 Cl▬ + H2O +
1O2
Superoxide anion Univalent reduction of O2 by hydrated electrons
Peroxide ion Two electron reduction product of O2
Hydrogen peroxide Radiolysis or photolysis of H2O.
Dismutation reactions.
OH● + OH● H2O2.
2O2●▬ + 2H+
H2O2
Hydroxyl radical Radiolysis or photolysis of H2O2, Fenton reaction
Fe2+ + H2O2 OH● + OH▬ + Fe3+
Ozone Polluted air and photochemical reactions like
O2 2O *
O2 + O* O3
Hypochlorous acid Enzymatic reactions
Peroxyl and alkoxy radicals
Organic hydroperoxide
Lipid peroxidation
Nitric oxide.
Nitrogen dioxide
Smoke
Peroxynitrite Smoke.
By the reaction O2●▬ + NO ONOO▬
1.3.2. Sources of free radicals in physiological systems
Both cellular metabolism and environmental factors serve as the source
of free radicals and other ROS in our body. Major biological sources of ROS
and RNS are given in Table 1.3
Solar energy
Introduction
27
Table 1.3 Biological sources of ROS and RNS (24-26).
ROS/RNS Endogenous sources
Singlet oxygen (1O2) Photosensitized oxidation & action of
myeloperoxidase (MPO) during phagocytosis
H2O + Cl▬ + H+ H2O + HOCl
HOCl H+ + OCl▬
OCl▬ + H2O2 Cl▬ + H2O +
1O2
Superoxide anion (O2●▬) Enzymatic reactions (eg; xanthine/XO),
autooxidation reaction of oxyhaemoglobin,
electron transport chains of mitochondria,
endoplasmic reticulam, phagocytosis etc.
Hydrogen peroxide (H2O2) Enzyme systems (eg; glycollate oxidase),
phagocytosis, dismutation of O2●▬ etc.
Hydroxyl radical (OH●) Radiolysis of H2O2, Fenton reaction etc.
Hypochlorous acid (HOCl) Action of MPO during phagocytosis.
Peroxyl radicals (ROO●) Lipid peroxidation
Nitric oxide (NO)
From vascular endothelial cells catalysed by nitric
oxide synthase.
Nitrogen dioxide (NO2) Combination of NO with O2 at body temperature
Peroxynitrite (ONOO▬) From vascular endothelial cells
1.3.3. Biological effects of free radicals
Aerobic organisms are exposed to a wide range of oxidants as part of
their metabolism. Free radicals and other reactive oxygen species are also
generated in the body as a result of stress, exercise, food habits, exposure to
pollution, radiation, pesticides etc. The free radicals formed in our body serve
important biological functions such as phagocytosis, cell signaling, apoptosis
etc. Free radicals and ROS are useful only when they are produced in right
MPO
Introduction
28
amounts and at right place and time. They in excess have the potential to
damage biomolecules such as proteins, lipids, and DNA.
1.3.3.1. Oxidative stress
Free radicals and other ROS are constantly formed in the human body.
Some is chemical accident, such as generation of hydroxyl radical by our
constant exposure to low levels of radiation from the environment and of
superoxide radical by leakage of electrons from electron transport chain.
Other production of these species is deliberate and beneficial. They can be
harmful when produced in excess and free radicals have been implicated in
the pathology of several human diseases including cancer, atherosclerosis,
rheumatoid arthritis and neurodegenerative diseases.
Human body is equipped with AO defense mechanisms to minimize
the effects of oxidants. Most cells can tolerate mild oxidative stress as they
have repair systems which recognize and replace oxidatively damaged
molecules. In addition cells may increase the AO defenses in response to the
stress. The disturbance in the balance between oxidants and AOs towards
oxidants is called ‘oxidation stress’. Oxidative stress causes oxidation of vital
biomolecules. Severe oxidative stress results in cell damage and death. It has
been implicated in numerous human diseases. Oxidative stress is thought to
be playing a major role in degenerative disorders such as CVD, rheumatoid
arthritis, neurodegenerative diseases, cancer and aging. The role of free
radical mediated reactions in degenerative diseases is tabulated in Table 1.4.
The role of free radical reaction in disease/ageing, toxicology, biology (Table
1.5) and in the deterioration of food (lipid peroxidation) has become an area
of intensive investigation.
Introduction
29
Table 1.4 Role of free radical reactions in biology (24-26).
ROS/RNS Deleterious Effect
Singlet oxygen Oxidation of lipids and proteins especially in the retina of
eye leading to cataract and age related molecular
degeneration (AMD).
Superoxide anion Oxidation of membrane lipids, reduction of cytochrome C
Hydrogen peroxide Inactivation of enzymes
Hydroxyl radical Membrane damage, DNA damage & strand breakage
Ozone Irritating eyes, nose and lungs, cross linking of proteins,
peroxidation of lipids, DNA damage etc.
Hypochlorous acid Damage to proteins, tissues& DNA, cell death caused by
cholesterol chlorohydrins.
Peroxyl radicals Lipid peroxidation, LDL oxidation.
Nitric oxide
Excess NO causes hypotension & insufficient amounts cause
hypertension.
Nitrogen dioxide Peroxidation of membrane lipids.
Peroxynitrite Damge to proteins, lipids and DNA
Table 1.5 Role of free radical reactions in diseases and ageing (24-26).
Disease/Disorder Free radical related event
Cardiovascular diseases Oxidative modification of LDL after initial damage
to vascular endothelium by other means.
Cancer DNA damage
Adult respiratory distress
syndrome (ARDS)
Damage to lungs
Radiation damage Damage to proteins, lipids and DNA.
Brain & spinal cord injuries Tissue damage after the injury
Keshan disease Mediated by oxidative stress
Cataract Oxidation of lens proteins
Ageing Damage to proteins, lipids, DNA and tissues.
Introduction
30
1.3.3.2. Lipid peroxidation: Oxidation of lipids have significant role in
deterioration of oils and fatty foods. Lipid peroxidation is an important patho-
physiological process occurring in numerous diseases and stress conditions
and results in a series of degradative processes affecting the organization and
function of cellular components. PUFA are more prone to lipid peroxidation.
The process begins when a free radical such as hydroxyl radical captures a
hydrogen atom from the methylene carbon in the polyalkyl chain of the FA.
Under aerobic conditions, a FA with an unpaired electron undergoes a
molecular rearrangement on reaction with oxygen, generate peroxy radical.
The peroxy radicals are highly reactive and can combine with other peroxy
radicals to alter the membranes. They can also capture hydrogen molecules
from adjacent FAs to form a lipid hydroperoxide thereby inducing the
propagation of lipid peroxidation. Thus, the peroxidation of unsaturated FAs
can induce the conversion of several FA side chains into lipid hydroperoxides,
thereby forming a reaction chain with the final product of lipid peroxidation
being malondialdehyde (MDA) (27). The major aldehyde product of lipid
peroxidation other than malondialdehyde is 4-hydroxy-2-nonenal (HNE).
Latter is weakly mutagenic but appears to be the major toxic product of lipid
peroxidation. The MDA thus formed, reacts with the free amino group of
proteins, nucleic acids and phospholipids to produce inter and intra molecular
1-amino-3-iminopropene (AIP) bridges. They can also produce a structural
modification of these biomolecules. The immune system recognizes these
MDA induced structures as non-self leading to an auto immune response (24).
Initiation
LH + OH● L● + H2O
Propagation
L● + O2 LOO●
LOO● + LH LOOH + L●
Introduction
31
Hydroperoxide decomposition
LOOH LO* MDA.
Moreover, the FA moieties of the plasma LDL can also be oxidized
during oxidative stress.
1.3.3.3. Beneficial Role of Free Radicals
Oxidation and production of radicals and ROS are an integral part of
metabolic processes. The free radicals may exert toxic effects but at the same
time, they are also essential for several biological functions. For example the
process of phagocytosis is facilitated by free radicals (27). One of its many
steps involves the production of superoxide and hydrogen peroxide in the
extra cellular space. Hydrogen peroxide is toxic to micro organisms either
directly by promoting several oxidations or indirectly by forming hydroxyl
radical or hypochlorite which in turn are more reactive.
ROS and RNS are known to play important roles in signal
transduction. The capacitation of spermatozoa by superoxide is an example
for the involvement of ROS in signal transduction. ROS may have beneficial
or detrimental effects on sperm fractions depending on the nature and
concentration of the ROS involved, as well as the moment and the location of
exposure. In particular, low concentrations of superoxide trigger this
phenomenon; where as excessive generation of hydrogen peroxide in semen
could be a cause for infertility. The RNS, nitric oxide, as an intracellular
messenger plays an important role in the nervous system, where it acts as a
neuromodulator and plays an important role in synaptic plasticity and long
term memory. In vascular system, it controls vasodialation and hence the
blood pressure. It is also associated with immune system (28).
Introduction
32
Programmed cell death (apoptosis) is needed for proper development
and to destroy cells that represent a threat to the integrity of the organism.
Apoptosis of a cell is initiated by the disturbance in balance between the
positive signals which help the survival of the cells (eg; growth factors for
neurons) and the negative signals (eg; increased level of oxidants within the
cell) (29). ROS are also used as therapeutic agents too. In photodynamic
therapy (PDT) the photosensitizer localized in the target tissue, is activated by
light to produce oxygen intermediates such as singlet oxygen that can destroy
target tissue cells. The easy access of skin to visible light and molecular
oxygen has led PDT successful in the treatment of basal cell carcinoma and
Bower’s disease. The most popular photosensitiser used in PDT is 8-
aminolevulinic acid.
1.3.4. Anti oxidants (AOs)
Free radicals and ROS are useful only when they are produced in right
amount, at right place and time. Otherwise, as mentioned, they will cause in
oxidative damage to the physiological systems. To protect the body from
harmful effects of free radicals and other oxidants all aerobic organisms are
endowed with powerful AO systems. These include physical defenses,
preventative and repair mechanisms and AO defenses (23). Human beings
and other living organism have developed powerful and complex AO
systems. AOs as defined by Halliwell and Gutteridge are group of substances
which, when present at low concentrations in relation to oxidizable substrates,
significantly inhibit or delay oxidative processes (30). Thus it is very
important to maintain equilibrium between pro oxidants and AOs. It can not
be solely maintained by endogenous AO system and therefore requires
external supply of AOs.
AOs intercept free radical mediated reactions and prevent oxidative
damage of cells. AOs function by a number of modes:
Introduction
33
Chain Breaking mechanism – A number of AOs are able to quench the free
radicals by donating hydrogen radicals or electrons and thus break the free
radical chain reactions. Thus AOs form new radicals with more stability
which leads to stable molecules (24,30,31). Such AOs are called primary
antioxidants. Examples include ascorbic acid and α- tocopherol. α- tocopherol
can stop lipid peroxidation by donating an electron to the peroxyl radical of
fatty acid there by stopping the propagation steps.
Scavenging initiating radicals - Certain AOs can react with the initiating
radicals, (or inhibiting the initiating enzymes) and such AOs are called
secondary AOs. Enzymes (eg: superoxide dismutase (SOD)) and the
compounds which inhibit enzymes such as xanthine oxidase (XO),
cyclooxygenase etc are typical examples. β-carotene is an efficient singlet
oxygen scavenger (24,30,31).
Metal chelation: Free metals can catalyse the formation of free radicals.
Certain AOs are able to chelate the transition metal and prevent them from
catalyzing free radical reactions. For example, albumin and ferritin are good
Fe2+ chelators. Some low molecular weight compounds such as polyphenols,
in addition to their ability to donate hydrogen, can also chelate transition
metal ions (24,30,31)
1.3.4.1. Classification of AOs
AOs are classified based on several criteria such as their origin,
solubility in lipid or water; physical and chemical characteristics etc. Based
on their origin, AOs can be classified into synthetic and natural AOs.
1.3.4.1a. Synthetic AOs.
In general synthetic AOs are compounds with phenolic structures of
various degrees of alkyl substitution. Synthetic AOs currently permitted for
Introduction
34
use in food include BHT, BHA, PG, TBHQ etc. BHA and BHT (Figure 1.6)
are fat soluble, volatile monohydric phenolics. These are extensively used in
packaged foods. TBHQ is regarded as the best AO for protecting frying oils
and fats. PG is sparingly water soluble and functions well in stabilizing
animal fats and vegetable oils. The use of synthetic antioxidants is becoming
increasingly restricted as many of them are reported to be carcinogenic and
this has resulted in an increased interest in the investigation for newer sources
of natural AOs.
Triphenyl radical
N
O
CH3
CH3
H3C
H3C
TEMPO DPPH radical
OH
CMe3Me3C
CH3
BHT
OH
OMe
CMe3
BHA
OH
OH
CMe3
TBHQ
O
O
OH
HO
HO
PG
Figure 1.6 Structures of some stable free radicals and synthetic AO
compounds
N∗
N
NO2
NO2
O2N
Introduction
35
1.3.4.1b. Natural AOs.
Natural AOs are in turn divided into two main classes: enzymatic and
non enzymatic AOs.
(i) Enzymatic AOs:
Cells have developed enzymatic systems which convert oxidants into
harmless molecules, thus protecting the organism from the deleterious effects
of oxidative stress. AO enzymes therefore have the capacity to lower the free
radical burden. They can inhibit the generation of free radicals during all the
stages of free radical reaction viz initiation, propagation and termination. The
various AO enzymes include;
Superoxide dismutase (SOD): SOD is one of the most effective intracellular
enzymatic AOs that catalyzes the dismutation of O2●▬ to O2 and to the less
toxic H2O2.
2 O2●▬ + 2H + SOD O2 +H2 O2
SOD is the first line in cell defense against oxidative stress. SOD exists in
several iso forms, differing in their nature of active metal centre and amino
acid composition, as well as their number of subunits, co-factors and other
features (23). In humans, there are three forms of SOD are reported: cytosolic
Cu-Zn SOD, mitochondrial Mn SOD, and extra cellular SOD. (32). SOD
destroys O2●▬ with remarkably high reaction rates by successive oxidation
and reduction of the transition metal ions at the active site (33).
Catalase:- Catalase is an enzyme present in the cells of plants, animals and
aerobic bacteria. It is located in the cytoplasm of RBC but compartmentalized
in the peroxisomes of the other cells. These enzymes catalyze the conversion
of H2O2 to H2O and molecular oxygen. Catalase also inhibits initiation phase
of free radical reaction (33).
Introduction
36
Glutathione peroxidase: There are 2 forms of the glutathione peroxidase
enzymes namely selenium independent (glutathione-S-transferase, GST) and
selenium dependent (GPx) glutathione peroxidase (33). GPx act in
conjunction with the tripeptide glutathione (GSH) present in the cells. GPx
converts lipid hydroperoxides (LOOH) to their corresponding alcohols (LOH)
with the simultaneous oxidation of GSH.
LOOH + 2GSH LOH + GSSG +H2O
GPx is also involved in the removal of H2O2
2GSH + H2O2 GPx GSSG + 2H2O
Thus GPx competes with catalase for H2O2 and is the major source of
protection against low levels of oxidative stress.
(ii) Non-enzymatic AOs:
Vitamin C: Vitamin C or ascorbic acid (AA) is a very important and
powerful AO that works in aqueous environments (23). It is found principally
in fresh vegetables and fruits and its deficiency is responsible for scurvy. AA
cooperates with Vitamin E to regenerate α- tocopherol from α- tocopheryl
radical in membrane and lipoproteins (34). AA is a di-acid as it has two
ionisable hydroxyl groups. At physiological pH, 99.9% of AA is present as
AScH▬, and only very small proportion as AScH2 (0.05%) and ASc2▬
(0.004%). AScH▬ is an electron donor AO and reacts with radicals to produce
resonance stabilized tricarbonyl ascorbate free radical (ASCH●). ASCH● has a
pK -0.86, and hence it is not protonated but it is present in the form of semi
dehydroascorbate radical (Asc●) a poorly reactive radical.
AA is known to protect membrane against oxidation. Recent in-vivo
and ex-vivo studies revealed that AA in plasma increases resistance to lipid-
peroxidation in a dose dependent manner, even in the presence of redox
active iron or copper and H2O2 (35). AA also prevents endothelial dysfunction
in chronic heart disease by inhibiting NO degradation (36). It neutralizes
GPx
Introduction
37
oxidants which are produced during macrophage activation by tobacco
smoke. Supplementation with AA showed a reduction in markers of oxidative
DNA, protein and lipid damage in a number of in vivo studies (37). However,
ascorbate is also shown to have pro oxidant properties and this effect of
ascorbate was attributed to the release of metal ions from damaged cells (38).
OO
OHHO
OHH
CH2HO
OO
OO
OHH
CH2
HO
L-ascorbic acid Dehydro ascorbic acid
Vitamin E: Vitamin E is a fat soluable vitamin that exists in eight different
forms such as- dα, dβ, dγ and dδ tocopherol and dα, dβ, dγ and dδ
tocotrienols. α-tocopherol is the most active form of vitamin E in humans and
is a powerful antioxidant (39). Tocopherol (Figure 1.3) inhibits lipid
peroxidation because they scavenge lipid peroxyl radicals much faster than
these radicals can react with adjacent fatty acid side chains or with membrane
proteins
α –tocopherol + lipid-O2 → α-tocopherol· + lipid –O2H
The –OH group of the tocopherol gives up its hydrogen atom to the peroxyl
radical, converting it to lipid peroxide. This leaves an unpaired electron on the
O. to produce a tocopheryl radical. Thus α-tocopherol breaks the chain
reaction of lipid peroxidation. The α-tocopheryl radical can be reduced to the
original α-tocopherol form by ascorbic acid (34)
Figure 1.7. Structures of L-ascorbic acid and dehydroascorbic acid
Introduction
38
Carotenoids: Carotenoids impart bright color to fruits, flowers and other
plant parts and are widely distributed in nature. β-carotene (Figure 1.3), the
most important carotenoid with wide distribution in plant kingdom is a
provitamin A. Carotenoids are considered as potent AOs. The presence of
lengthy conjugate double bond system enables carotenoids to act as effective
singlet oxygen quenchers.
1O2 + β-carotene 3O2 +β-carotene
Thus β-carotene and other carotenoids in plants, serve as AOs and help to
scavenge singlet oxygen that can be generated by the interaction of light with
the plant pigment chlorophyll. β-carotene may also exert other AO effects
such as scavenging of free radicals in lipid systems (40).
Thiol AOs
Glutathione: The tripeptide glutathione (γ -L-glutamyl -L-cysteinyl- L-
glycine) is the major thiol AO. It is present in large quantities in organs
exposed to toxins such as the kidney, the liver, the lungs and the intestine, but
very little is found in body fluids. The reduced form of glutathione is GSH
(glutathione) and oxidized form is GSSG (Glutathione disulphide) tripeptide.
The AO capacity of glutathione and other thiol compound is due to sulphur
atom which can easily accommodate the loss of electrons. Also the life time
of sulfur radical species formed (GS●) is significantly longer than many other
radicals generated during the stress.
GSH + R● GS● + RH
GS● + GS● GSSG
GSH also serves as a cofactor of several detoxifying enzymes such as
glutathione peroxidase and glutathione transferase against oxidative stress.
GSH participate in amino acid transport through the plasma membrane. GSH
is an effective scavenger of hydroxyl radical and singlet oxygen. GSH is also
able to regenerate vitamin C and E back to their active forms. GSH in the
Introduction
39
nucleus helps to maintain the redox state of protein sulfahydryls that are
necessary for DNA repair and expression (41,42).
Thioredoxin and glutaredoxin systems: The thioredoxin system comprises
of thioredoxin reductase (TS) and thioredoxin (TrX) a small multi functional
disulfide containing redox protein NADPH. It reduces the disulfide bond of
several proteins and oxidized GSH to thiol groups. The thioredoxin system
thus regulates the activity of proteins such as the transcription factors NFkB
and AP-1 (41). Thioredoxin can also reduce other compounds especially lipid
hydroperoxides. Trx and TR contribute to maintain the redox status in the
plasma by acting as electron donors for the blood plasma peroxidase (43). The
mechanism of action of the glutaredoxin system is similar to the thioredoxin
system and the former is composed of glutaredoxin (Grx), glutaredoxin
reductase (GR) and NADPH. It also needs the presence of glutathione (44). It
was suggested that it plays an important role in the intra cellular balance
between GSH/GSSG and in the glutathionylation of proteins such as carbonic
anhydrase (41, 44).
Taurine and hypotaurine: Taurine and its precursor hypotaurine are β amino
acids derived from cysteine metabolism (45). Hypotaurine has shown to have
the capacity to scavenge hydroxyl radical and to inhibit lipid peroxidation.
Taurine supplementation has been shown to decrease lipid peroxidation and it
also protects the heart during reperfusion post ischemic (46).
HO
S
O
O
NH2
SNH2
O
HO
Taurine Hypotaurine
Figure 1.8 Structures of taurine and hypotaurine
Introduction
40
α-Lipoic acid: α-Lipoic acid or thiothic acid is a disulphide derivative of
octanoic acid. α-Lipoic acid is both water and fat soluble and hence is widely
distributed in both cellular membrane and the cytosol. It exists in cells as
lipoamide which is covalently bound to different cytoplasmic protein. Lipoate
is reduced to dihydrolipoate by glutathione reductase, thioredoxin reductase
or dihydrolipoamiae dehydrogenase. Both α-Lipoic acid and dihydrolipoic
acid are powerful AOs (47). Their AO functions involve quenching of ROS,
regeneration of endogenous and exogenous AOs involving vitamin C and E
and glutathione, chelation of redox metals, and repair of oxidized proteins
(48).
1.3.5. AO related medicinal properties of phytochemicals
The AO nature of phytochemicals is largely responsible for their anticancer,
cardioprotective, immunomodulatorty, neuroprotective, and antimicrobial
properties (49- 52). Phenolic compounds and fruit extracts have been reported
to have positive effects on cancer, CVD, immune disorders, microbial
infections, neurodegenerative disease and viral infections (53-59). Vitamin C,
tocopherols and phenolics are the major classes of phytochemicals with AO
properties.
SS
COOH
Figure 1.9. Lipoic acid
Introduction
41
1.3.5.1. AO activity of phytochemicals
The AO activity of phytochemicals is related to a) scavenging free radicals, b)
chelating transition-metals involved in free-radical production and c)
inhibiting the enzymes participating in free-radical generation (24,30,31,60-
62). The free radical scavenging activity of phenolic compounds is generally
attributed to their ability to donate a hydrogen atom to reduce ROS radicals
(63). In doing so, the phenolic compounds (ArOH) are converted to oxidized
phenoxy radicals (ArO•) that are stable due to resonance-stabilized
delocalization of the unpaired electron over the aromatic ring (61,64). For
example, the reduction of peroxyl and hydroxyl radicals by phenolic
compounds can be represented as follows:
ROO• + ArOH → ROOH + ArO•
HO• + ArOH → HOH + ArO•
The phenoxy radical intermediates are relatively stable and therefore further
oxidation reactions are not easily initiated. Non-radical products may also be
formed by the coupling of ROS radicals with phenoxy radicals (63).
ROO• + ArO• → ROOArO
Phenolic compounds may also enhance the AO activity of non-
phenolic AOs by regenerating the oxidized forms of these compounds. For
example, phenolic compounds have been reported to regenerate ascorbic acid
from its oxidized form (65). The synergistic AO interactions between
flavonoids and α-tocopherol in vitro have been demonstrated by Pedrielli and
Skibsted (66). (+)-catechin, (-)- epicatechin and quercetin are found to
enhance the induction period for oxidation of α-tocopherol and results in
longer and increased inhibition of oxidation. The regeneration of α-tocopherol
from its oxidized form through the hydrogen atom transfer between
flavonoids and tocopherol was suggested as the mechanism for this action.
Metal chelating effects of phenolics due to their catechol, galloyl, or
1,3 positioned hydroxyl and carbonyl moieties also imparts secondary AO
Introduction
42
effects by inactivating metals (31, 61). Phenolic acids and flavonoids have
been shown to complex with iron (67) and copper ions (68) to provide
secondary AO effects.
1.3.5.2. Medicinal Effects of Phytochemicals
There are numerous in vitro and in vivo studies that have indicated the
potential medicinal benefits of phenolic compounds. Excellent reviews on this
topic have been published (50, 69-73)
Oxidation of LDL is a key event leading to the formation of
atherosclerotic plaques (74). It is believed that ROS as well as enzymes, such
as lipoxygenase and myeloperoxidase, are involved in the oxidation of LDL
(71,75). Some studies have shown the role of dietary phenolics in preventing
CVD through their AO effects (69,76,77). Flavonoids have also been reported
to exert positive effects on the cardiovascular system through modulation of
the nitric oxide synthase system (78,79). The anthocyanins extracted from
bilberry (Vaccinium myrtillus) juice have been reported to protect the vascular
system in vivo by increasing the permeability of capillary blood vessels (80).
Phenolic compounds in red wine are hypothesized to be the beneficial
components responsible for inhibiting the oxidation of LDL and thus,
providing protection against atherosclerosis (81). Resveratrol, a stilbene has
been reported to exhibit AO (82), cardioprotective (83) and anti-inflammatory
properties (84). However, not all epidemiological studies favor a protective
effect of dietary phenolic compounds against CVD (84,85).
Consumption of green vegetables and fruits found to reduce greatly the
risk of cancer (86). Yen and Chen showed that aqueous extracts of black,
green, Oolong and Pouchong tea inhibit > 90% of the mutagenicity of a
selected number of chemicals toward S. typhimurium TA98 and TA100 (87).
Epigallocatechin gallate (EGCG), a phenolic component of green tea, has
Introduction
43
been found to reduce the incidence of chemically induced tumors in the
esophagus, liver, lungs, skin and stomach of experimental animals (88).
EGCG was also reported to possess effective AO properties and can provide
protection in vitro against both peroxyl radical and hydroxyl radical induced
oxidation of DNA (89). Anthocyanins have been shown to have stimulatory
effect on the secretion of tumour necrosis factor-α in macrophages in vitro
(90) which are responsible for cytostatic and cytotoxic activities on malignant
cells (91). Citrus flavonoids, in particular, flavanones have been shown to
display in vitro anticarcinogenic and antiallergenic (92) activities. Hibasami et
al; reported that catechin-rich persimmon induced apoptosis in human
lymphoid leukemia cells (93). Wang et al; reported that lingonberries had
potent free radical scavenging and apoptosis inducing activities (94).
Other studies have shown that resveratrol from grape can inhibit
cellular events associated with carcinogenesis including tumour initiation,
promotion and progression (95). In vivo studies have indicated that
resveratrol has anticancer effects against intestinal (96), colon (97) and skin
cancer (73). In contrast, some other studies reported no anticancer effect and
even carcinogenic properties at high dosage levels of resveratrol (98,99).
Furthermore, it is noted that the very low concentration of resveratrol in red
wine (0.3 to 2.0 mg/L) makes the attribution of protective effects to this
compound unlikely (100,101). Combination of resveratrol and quercetin
synergistically induce apoptosis in human leukemia cells (86). Other studies
have suggested that caffeic acid and some of its esters possess antitumour
activity against colon carcinogenesis (102,103).
Lignans are recognized as phytoestrogens due to their estrogen
moderating activity (104). There is evidence that lignans from flaxseed can
reduce mammary tumor size and number in rats (105,106). Genistein, a soy
derived ‘phytoestrogen’ has the potential as chemotherapeutic agents capable
Introduction
44
of inducing apoptosis by inhibiting DNA topoisomerase and angiogenesis
(107) or suppressing tumor promoting proteins such as COX-2 (108).
Correlation between the consumption of phenolic compounds and improved
health has been reported in epidemiological studies (73, 109)..
Drug phytochemicals interactions also result in varying effects on
cancer treatment. For example quercetin has been found to enhance the
activity of anticancer drugs such as triazofurin and carboxytriazole on human
cancer cells (110,111). The apoptotic inducing activity of EGCG on lung
cancer PC-9 cells have been synergistically enhanced by other
chemopreventive agents such as sulindac and tamoxifen (112,113). Many
cancer drugs are synthesized on the basis of novel structures provided by
natural products. Many natural products such as taxanes, vinca alkaloids,
podophyllotaxins and camptothecins are served as chemotypes for anticancer
drugs (114)
It has been proposed that the phenolic AOs that are consumed through
foods, medical foods and/or dietary supplements seldom reach levels in vivo
that are sufficient to function as effective AOs. According to Hensley et al;
the level of external phenolic AOs can range from concentrations of high nM
to low µM concentrations in cells and blood plasma (62). These authors
suggested that the minimum concentration for effective free radical
scavenging activity of these AOs, however, is in the medium to high µM
concentration range (i.e.50 to greater than 100 µM). In spite of the limited AO
levels in foods, they noted physiological benefits of phenolic free radical
scavengers in inhibiting diseases associated with oxidative stress. It was
proposed that the protective effect of many phenolic AOs, however, may be
largely due to inhibition of enzyme systems or signal transducers that are
responsible for the generation of ROS. In support of this hypothesis for in
vivo AO activity, others have reported that phenolic compounds show marked
Introduction
45
inhibition of enzymes responsible for the generation of ROS such as nitric
oxide synthase (78,79,90), tyrosine kinase (115) and xanthine oxidase (116).
It is important to note that under certain conditions phenolic AOs may act as
promoters of free radicals and thus, act as pro-oxidants. Such conditions have
been reported to include high concentrations of the phenoxy radicals resulting
from low concentrations of synergistic AOs or a lack of reducing enzymes to
regenerate the AO from its phenoxy radical state (117).
Tea phenolic compounds have also been also reported to exhibit some
other physiological activities. Green tea phenolic components inhibit
intestinal uptake of glucose through rabbit intestinal epithelial cells and thus
may contribute to the reduction of blood glucose levels (118). Various in vitro
studies have been reported that flavonols inhibit many pathogenic
microorganisms including Vibrio cholerae (70), Streptococcus mutans (119)
and Shigella (120). PA rich extracts from grape seeds also display anticataract
activity in rats and the inhibition of cataract progression was suggested to be
due to the AO activity of the grape seed PA (121). In other studies, grape seed
extracts were found to possess antiulcer properties in rats (99). Caffeic acid is
known to selectively block the biosynthesis of leukotrienes, components
involved in immunoregulation diseases, asthma and allergic reactions (122).
Caffeic and ferulic acid derivatives have been suggested to be potential
protective agents against photooxidative skin damage (123). Quercetin has
been reported to be a potent inhibitor of human immunodeficiency virus
(HIV)-1 protease (58). This compound has also been shown to decrease the
infectivity of herpes simplex virus type I, poliovirus type I and parainfluenza
virus in vitro (124).
Introduction
46
1.4. Sea Buckthorn (Hippophae rhamnoides)
Introduction
47
Sea buckthorn (SB) (Hippophae rhamnoides), is an economically
important medicinal plant belonging to Elaeagnaceae family. It grows in cold
regions of Asia, Europe, and North America. The distribution ranges from
Himalayan regions including India, Nepal, Bhutan, Pakistan and Afghanistan,
to China, Mongolia, Russia, Kazakstan, Hungary, Romania, Switzerland,
Germany, France and Britain, and northwards to Finland, Sweden and
Norway (125-128). The wide distribution of SB is reflected in its habit-related
variation not only in morphology, yield, growth rhythms and cold hardiness,
but also in berry related characters such as fresh weight, chemical and sensory
attributes (126, 129). SB is known in different languages as Chharma (Hindi),
Shaji (Chinese), and Sanddrome (German). In Greece, SB leaves and twigs
are added in fodder to gain weight and shiny coat for the horse. This gives the
name to the bush ‘Hippophae’. ‘Hippo’ means horse and ‘phoas’ means
shine. SB includes 5 or more species namely H. rhamnoides, H. salicifolia, H.
tibetana, H. neurocarpa and H. goniocarpa. In these H .rhamnoides is the
most common and important species and is further classified into nine
subspecies (ssp.) namely rhamnoides, fluviatilis, carpatica, caucasica,
mongolica, yunnanensis, gyatsensis, Sinensis, and turkestanica (130).
The SB is a hardy deciduous shrub with narrow leaves, reaching 2 to 4
meters in height and develops a tree-like appearance since usually only the
upper buds sprout and branch (131). SB is adapted to cold, drought as well as
saline and alkaline soils. Consistent with other members of the Elaeagnaceae
family, SB is also a nitrogen fixer (132). Its vigorous production of vegetation
and its strong and complex root system with nitrogen-fixing nodules make SB
an optimal pioneer plant for water and soil conservation in eroded land. Over
one million SB seedlings reported to have been planted in the Canadian
Prairies since 1982 as a part of land reclamation program (133). The shrub is
also planted in British Columbia, Newfoundland and Quebec (127).
Introduction
48
In summer, plentiful round yellow-orange berries cover the female
plants. SB berries consist of a fairly tough skin and juicy pulp enveloping a
small, hard, oval seed. Ripe berries are orange-red in color and have a
diameter of 10–15 mm and a soft outer fleshy tissue and hard seed. A natural
SB can yield 750 to 1500 kg fruit/ha (134). SB fruit has attracted considerable
attention, mainly for its medicinal value and great economic potential.
The fruit and leaves have been used for more than one thousand years
in traditional medicines in India, China, Mongolia and Tibet (135). In Asia
and Russia fruit and seed extracts have been used for the treatment of burns,
cancer, CVD, gastric ulcers and oral inflammation (136,137). SB oil is
approved for clinical use in hospitals in China and Russia, where, in 1977, it
was formally listed in the Pharmacopoeia (134, 135). More than ten different
drugs have been developed from SB in these countries (134).
1.4.1 Sea Buckthorn in India
In India, SB is found in Himalayan regions of Himachal Pradesh,
Ladakh (J&K), Uttaranchal, Sikkim and Arunachal Pradesh. SB cultivation in
India is mainly concentrated in the cold deserts of Trans-Himalyas (Ladakh,
Lahaul and Spiti) at an altitude from 2500 m to 4500 m. Indian Himalayas
host world’s second or third largest area under SB (30,000 ha). In Ladakh it
grows approximately 12,000 hector area and covers major part of the forest
area in the region The SB-growing areas in the Trans-Himalaya host unique
geoclimatic conditions of high altitude coupled with extreme temperature
variations (–30 to 30°C), low precipitation, low oxygen in air and arid soil.
Mainly three species of SB viz; H. rhamnoides var. turkestanica, H. salcifolia
and H tibetana are found in India. Among these H. rhamnoides var.
Turkestanica is the most common in India. The SB-growing areas in the
Trans-Himalaya host unique geo-climatic conditions of high altitude coupled
Introduction
49
with extreme temperature variations (–30 to 30°C), low precipitation, and low
oxygen in air (138).
1.4.2. Chemical Composition of SB berries
SB berry is recognized for its nutritional benefits, being rich in amino
acids, carbohydrates, organic acids, protein, vitamins and phenolic
compounds (131). A number of studies have shown that the chemical
composition of the fruit varies greatly according to the species, and climatic
and geological conditions of the areas where the plant is grown
(126,139,140).
The moisture content of SB fruit has been reported to range from 74.0
to 86.7% (140,141). Carbohydrate content in the fresh berries of Chinese
origin varies between 8.0 – 14.0% with glucose and fructose as the major
carbohydrates and are present in approximately equal amounts (142). The
protein content of the fruit varies with the variety and geographical location
and is reported to vary from 0.79 to 3.11% on fresh weight basis (143).
SB is reputed to be an excellent source of vitamin C, although a wide
concentration range (2 to 2500 mg/100 g juice) has been reported
(131,126,144, 146). SB berries are highly acidic in nature. Beveridge et al;
reported an average pH of 3.13, and a titratable acidity of 1.97% as malic acid
in juice extracted from berries grown in Canada (147). Malic acid was the
major organic acid reported in Finnish SB (ssp. rhamnoides) with minor
quantities of citric and tartaric acid (139).
Yang and Kallio determined the oil content of Chinese and Finnish SB
varieties. The whole berry oil content of Chinese SB was 2.1% and 1.7% of
the seed and pulp/peel respectively (129). The whole berry oil content of
Finnish SB was reported to be 3.5% with an oil content of 11.3% of the seed
Introduction
50
and 2.8% of the pulp/peel. Yang and Kallio also published an excellent
review on the composition, nutritional effects, and industrial application of
SB lipids (148).
The oil content and fatty acid (FA) composition of berries from two
subspecies of SB (H. rhamnoides L.) were investigated by Yang et al (129).
The berries of ssp. rhamnoides contained a higher proportion of oil in seeds
(11.3% vs 7.3%), berries (3.5% vs 2.1%), and seedless parts (2.8% vs 1.7%)
than the berries of ssp. sinensis. Linoleic (18:2 n-6) and linolenic acids (18:3
n-3) comprise about 70% of seed oil FA. Palmitoleic acid (16:1 n-7)
comprised 12.1-39.0% of oil in pulp/peel. More linoleic acid (40.9% vs
39.1%) and less linolenic acid (26.6% vs 30.6%) are found in the seed oil of
ssp. sinensis than those in the seed oil of ssp. rhamnoides. Palmitoleic acid is
practically absent in seed oils.
Ozerinina and coworkers investigated the composition and structure of
the triacylglycerols (TAGs) of SB seeds belonging to Russian varieties with
the aid of lipase hydrolysis (149). Various SB (H. rhamnoides L.) climatypes
are classified into two classes based on the composition, structure, and
biosynthetic pattern of TAGs of their fruit mesocarp oil; (1) Siberian, Central
Asian, and Baltic, and (2) Caucasian. Group 1 oils contain predominantly
monosaturated-diunsaturated (SU2) TAG, which include C16:0 and C16:1
fatty acid (FA) residues. Berry mesocarp oil of SB of the Caucasian climatype
includes mainly disaturated-monounsaturated (S2U) and SU2 TAGs rich in
the residues of C16:0 and C18:1 isomers. As for the seeds, the climatypes
studied here practically do not differ from each other in the FA composition
of their TAGs (149, 150).
HPTLC and GC profiling of FA in total and individual polar lipids
separated from carotenolipoprotein complexes in SB berries were reported.
Introduction
51
The polar lipids included 61% phospholipids and 39% galactolipids, which
contained mainly 16:0, 16:1 (n-7), 18:1 (n-9), 18:1 n-7 and 18:2 (n-9, 6) FA.
Almost all polar lipids showed high ratios of 16:0/16:1 and 18:1 (n-9)/18:1 (n-
7), and higher quantities of 18 carbon unsaturated FA than of the saturated
analogue. Galactolipids were shown to be rich in 18:1 (n-9) and 18:3 (n-9, n-
6, n-15) FA, while phospholipids contained higher concentrations of 16:0 and
18:1 (n-9) FAs (151).
Application of capillary supercritical fluid chromatograph (SPC),
combined with a triple-quadruple mass spectrometer (MS) via a liquid
chromatography-atmospheric pressure chemical ionization (LC-APCI)
interface for the analysis of SB berry oil TG has been demonstrated by
Manninen et al (152). Zadernowski et al; showed that lipase activity was
reduced by 40-70% and 13-50% by lipophilic and hydrophilic-EtOH extracts
of SB berries of Poland origin (153).
The total sterol content was found to range from 1200 to 1800, 240 to
400 and 340 to 520 mg/kg in the oils from seeds, fresh pulp/peel and whole
berries, respectively (154). Sitosterol constituted 57 to 76% and 61 to 83% of
the seed and pulp/peel sterols, respectively. Salenko et al reported the
presence of β-Sitosterol, 24-methylene-cycloartanol, citrostadienol, and uvaol
in the unsaponifiable part of a pentane extract of the fruit pulp of common SB
(155). Phytosterols in SB (H. rhamnoides L.) seed oil extracted by cold
pressing, hexane, and supercritical carbon dioxide (SC-CO2) were identified
by GC-MS and FID and reported that sitosterol and δ-5-avenasterol were,
quantitatively, the most important phytosterols (156).
The carotenoid content of SB fruit varies with the geoclimatic
conditions but typically ranges from 30 to 40 mg/100 g fruit (131) with β-
carotene accounting for approximately 45% of the total carotenoids. HPLC
Introduction
52
quantification of free tocopherol content in whole berries of six SB cultivars
grown in northeastern Poland and Belorussia was reported (157). The total
free tocopherol content in oil from whole berries was 101–128 mg/100g of oil
with α-tocopherol as the predominant. α- and δ-Tocopherols constituted 62.5–
67.9% and 32.1–37.5% of total tocopherol, respectively, and only traces of γ-
tocopherol were detected in the oil. Green berries contained a marked amount
of γ-tocopherol, but its content rapidly declined to traces when the color of
berries turned from green to olive-yellow.
The total phenolic content of SB fruit was reported to range from 114
to 244 mg/100 g fruit (158). These authors reported a strong positive
correlation between the AO capacity of the fruit and its total phenolic and
ascorbic acid contents. Phenolics, including flavonols, flavones, phenolic
acids, PAs and hydrolysable tannins are reported as the major contributors to
the biological properties like AO activities of SB berries and leaves
(144,157).
The flavonoid content in the leaves and fruit of SB has been reported to
range from 310 to 2100 mg/100 g dried leaf and 120 to 1000 mg/100 g fresh
fruit, respectively (141). Analytical and preparative countercurrent
chromatographic separation of flavonoid constituents from crude ethanol
extract of SB dried fruits has been demonstrated (159,160). Preparative
isolation and purification of flavonoids and protocatechuic acid from SB juice
concentrate by high-speed counter-current chromatography was also reported
(161). Zhang et al; optimized the conditions for the simultaneous
determination of quercetin, kaempherol, and isorhamnetin in
phytopharmaceuticals containing SB by HPLC with chemiluminescence
detection (162). HPLC-DAD analysis of flavonoids in SB leaves has been
described by Zu et al. and reported the presence of catechin, quercetin,
isorhamnetin and rutin (163).
Introduction
53
Jeppsson et al; investigated the variations in contents of kaempherol,
quercetin and L-ascorbic acid in the berries during maturation using HPLC.
The content of ascorbic acid and quercetin decreased over the time for the five
cultivars studied, whereas kaempherol increased during maturation (125).
Rosch et al; investigated the phenolic composition of SB fruit juice by HPLC-
DAD and ECD (164). Flavonols are found to be the predominating
polyphenols while phenolic acids and catechins present in minor amounts.
Quantitatively isorhamnetin-3-O-glycoside is the predominant and is a poor
radical scavenger as shown by ESR. Phenolic compounds such as quercetin 3-
O-glycosides, catechins, and hydroxybenzoic acids with a catechol structure
exhibited good AO capacities. These phenolic compounds account for less
than 5% of the total AO activity of the filtered juice and ascorbic acid is
shown to be the major AO in SB juice.
In another study isolation of 4 flavonol glycosides from SB pomace
(H. rhamnoides) by Sephadex LH-20 gel chromatography and semi-
preparative HPLC was reported. The occurrence of the major flavonol
glycoside kaempferol-3-O-sophoroside-7-O-rhamnoside in SB is also
reported. Most of the compounds identified were 7-O-rhamnosides of
isorhamnetin, kaempferol, and quercetin, which exhibit different substitution
patterns at the C-3 position, mainly glucosides, rutinosides, and sophorosides.
In addition, numerous flavonol glycosides were detected lacking a sugar
moiety at C-7. Finally, eight flavonol derivatives were identified that are
acylated by hydroxybenzoic or hydoxycinnamic acids (165). Recently, Chen
et al; reported the development of a HPLC fingerprint method for
investigating and demonstrating the variance of flavonoids among different
origins of SB berries (166). Thirty-four samples were analyzed including 15
H. rhamnoides ssp. sinensis samples, 7 H. rhamnoindes ssp. yunnanensis
samples, 5 RW H. rhamnoides ssp. wolongensis samples, 4 NS H. neurocarpa
Introduction
54
ssp. stellatopilosa samples and 3 TI H. tibetana samples and 12 flavonoids are
identified from HPLC chromatograms.
Rosch et al; reported the identification of monomeric flavonols and PA
from SB (H. rhamnoides) pomace (167). Five dimeric PA are identified by
HPLC-ESI-MS/MS and by acid catalyzed cleavage in the presence of
phloroglucinol. Nine trimeric PA are tentatively identified by HPLC-ESI-
MS/MS in the Sephadex fractions. The isolated flavan-3-ols and
proanthocyanidins are potent in scavenging Fermy's salt, a synthetic free
radical. They possess antioxidant capacities that are higher or comparable to
that of ascorbic acid or trolox. On comparing the antioxidant capacities of
monomeric flavan-3-ols and dimeric PA, no significant influence from the
degree of polymerization (DP) was observed.
In another report monomeric flavan-3-ols, and dimeric and trimeric
PAs are fractionated from an extract of SB (H. rhamnoides) pomace by
Sephadex LH-20 gel chromatography and subjected to AO assays. The
oligomeric fraction accounted for 84% of the total PA and 75% of the total
AO activity of the SB pomace extract. Quantitative HPLC, NMR and MS
investigations demonstrate (+)-gallocatechin as the predominating subunit in
the oligomeric fraction and the majority of the flavan-3-ol subunits possessed
a 2,3-trans configuration. The oligomers consisted mainly of prodelphinidin
subunits whereas procyanidins were present in smaller amounts, indicating a
very uncommon composition of the SB PA. The mean DP of the oligomeric
PA is between 6 and 9 (165). In a recent report 4 monomeric flavan-3-ols
(catechin, epicatechin, gallocatechin and epigallocatechin), along with 2
dimeric procyanidins, (catechin-(4α-8)-catechin and catechin-(4α-8)-
epicatechin), are isolated from seeds. Polymeric PAs are also fractionated and
their chemical constitutions studied by acid-catalysed degradation in the
presence of toluene- α -thiol. The results showed highly heterogeneous
Introduction
55
polymers, with catechin, epicatechin, gallocatechin and epigallocatechin as
the constituent components of both the extension as well as the terminating
units. The mean DP is 12.2 and the proportion of prodelphinidins was 81.2%
(168).
In a recent report analysis of AO compounds such as trans-resveratrol,
catechin, myricetin, quercetin, p-coumaric acid, caffeic acid, L-ascorbic acid,
and gallic acid in six different varieties of SB berries (SB varieties:
"Trofimovskaja (TR)," "Podarok Sadu (PS)," and "Avgustinka (AV),") is
published. Trans-Resveratrol, catechin, ascorbic acid, myricetin, and
quercetin were found in all SB extracts. The biggest average AA content was
found in TR (740 mg/100 g of dried berries). The same varieties gave the
highest quercetin content 116 mg/100 g of dried berries) (169).
Rosch et al; reported the amount of total GA and ProCA in SB berries
from Finland by HPLC analysis (164). Zadernowski et al; found that the
phenolic acid composition in SB berries ranged from 3570 to 4439 mg/kg on
dry weight basis. They tentatively identified 17 phenolic acids in the fruit
with salicylic acid accounting for 55 to 74% of the total. The phenolic acids in
the fruit were mainly in their esterified and glycosylated forms, whereas the
maximum free phenolic acids content was 2.3% (170).
The reported chemical composition of SB berries varies considerably.
This may be because of different origins/subspecies, the climate and
geographical conditions of growing areas, and agronomic practices (129). A
systematic mapping of the chemical composition of SB berries of different
varieties and origins is still lacking.
Nutritional qualities of different varieties of SB berries belonging to
different geoclimatic conditions are compared in terms of their chemical
Introduction
56
composition by several authors. Kallio et al; evaluated the vitamin C,
tocopherols, and tocotrienols contents in berries of wild and cultivated SB (H.
rhamnoides L.) of different origins and harvesting dates. Wild berries of ssp.
sinensis, native to China, contained 5-10 times more vitamin C in the juice
fraction than the berries of ssp. rhamnoides from Europe and ssp. mongolica
from Russia (4.0 – 13.0 v/s 0.02-2.0 g/L juice). For bushes cultivated in
southwest Finland, the best berry harvesting date for high vitamin C content
was the end of August. The seeds of ssp. sinensis contained less tocopherols
and tocotrienols (average 130 mg/kg) compared with seeds of ssp.
rhamnoides (average 290 mg/kg) and mongolica (average 250 mg/kg). The
fruit flesh of sinensis berries had contents of tocopherols and tocotrienols 2-3
times higher than those found in the other two subspecies (120 mg/kg vs 40
mg/kg in rhamnoides and 50 mg/kg in mongolica). The total content of
tocopherols and tocotrienols in the soft parts of the berries reached the
maximum level around early- to mid-September, whereas the content in seeds
continued to increase until the end of November (145).
Berries and seeds of two subspecies (ssp. sinensis and mongolica) of
SB (H. rhamnoides L.) have been compared in terms of TAG,
glycerophospholipids (GPL), tocopherols, and tocotrienols. The study shows
that the berries of ssp. mongolica contained less oleic acid (4.6 vs 20.2%) and
more palmitic (33.9 vs 27.4%) and palmitoleic (32.8 vs 21.9%) acids in TAG
than those of ssp. sinensis. The proportions of linoleic acid (32.1 vs 22.2%, in
berries; 47.7 vs 42.7%, in seeds) and palmitic acid (21.1 vs 16.4%, in berries;
17.0 vs 14.1%, in seeds) in GPL are higher in ssp. mongolica than in ssp.
sinensis, and vice versa with oleic acid (4.3 vs 18.5% in berries, 10.0 vs
22.2% in seeds). A higher proportion of linolenic acid is also found in the
GPL of ssp. sinensis berries (16.2 vs 10.1%). Tocopherols constitute 93-98%
of total tocols in seeds, and α-tocopherol alone constitutes 76-89% in berries.
The total contents of tocols vary within the ranges of 84-318 and 56-140
Introduction
57
mg/kg in seeds and whole berries, respectively. The seeds of ssp. mongolica
are a better source of tocols than those of ssp. sinensis (287 vs 122 mg/ kg, p
< 0.001) (171).
Tsydendambaev et al; evaluated the changes in the quantitative
composition of TAGs in maturing SB (H. rhamnoides L.) seeds by lipase
hydrolysis and reported that as a whole, the rate of synthesis of separate TAG
classes increased in proportion to both their unsaturation and relative content
(weight percent) in total TAGs (170). Essential oil and FA composition of the
SB fruits (H. rhamnoides) L. Turkey was reported by Cakir (173).
TAG of seeds, berries, and fruit pulp/peel of different subspecies of SB
(H. rhamnoides) has been analyzed by MS and tandem mass spectrometry
(MS/MS). The study shown that the seeds contained mainly TAG with acyl
carbon number (ACN) of 52 with 2-6 double bonds (DB) (20-30%), and TAG
of ACN 54 with 3-9 DB (70-80%). In the pulp/peel fraction, the major TAG
were species with ACN:DB of 48:1 to 48:3 (19-49%), 50:1 to 50:4 (31-41%),
and 52:1 to 52:6 (9-19%). Ssp. sinensis differed from ssp. mongolica and
rhamnoides by having a higher proportion of TAG of ACN 52 (27% vs. 21%
and 22%) and a lower proportion of ACN 54 (71% vs. 79% and 78%) in seed
TAG. Seed TAG of ssp. mongolica contained a higher proportion of more
unsaturated species compared with those of the two other subspecies. Berry
TAG of ssp. mongolica had the highest proportion of molecular species of
ACN 48 due to the higher proportion of palmitic and palmitoleic acids and the
lower seed content of the berries. Overall, palmitic acid favored the sn-1 and
sn-3 positions. The order of preference of unsaturated FA for the sn-2 position
depended at least partially on the FA combination of TAG. Seed TAG of ssp.
mongolica contained a higher proportion of linolenic acid in the sn-2 position
than those of ssp. sinensis. In berry TAG, ssp. mongolica had the highest
proportions of palmitoleic and linoleic acids in the sn-2 position, and the
Introduction
58
lowest proportion of oleic/cis-vaccenic acid in the sn-2 position, among the
three subspecies (174).
Abid et al; recently reported the physico-chemical characteristics and
FA profiles of seed and pulp oils of SB (H. rhamnodes L) wildly grown in
Northern Areas of Pakistan (Skardu). Pulp oil with 10.0% yield has palmitic
acid (34.5 %) and almitoleic acid (33.4 %) as major FA. Oleic acid (22.1 %),
linoleic acid (29.6 %) and linolenic acid (23.4 %) are the major FA in seeds
oil (yield 4.5%) (175).
1.4.2. Processing of SB berries
Being a good source of bioactive phytochemicals, SB berries have
been processed by hundreds of industries in China and Russia for
nutraceutical and cosmaceutical products. Reports describing the processing
of SB berries are rather limited. Beveridge et al and Zeb et al 2004
extensively reviewed the various processing techniques of SB berries and
applications of products (127, 176). Both authors tabulated the available
compositional data for the main products to form comprehensive sources of
information on the manufacture and composition of SB products. Juice, pulp
oil, seed oil, cream and pigments are the main commercial products from SB
berries. Normally the processing begins with the harvesting of berries. The
diseased and damaged berries and stems, leaves and other debris are removed
as a part of cleaning. Washing the berries with luke warm water or with mild
detergents or wetting agents are suggested to increase the juice yield. Pressing
techniques such as screw pressing, cloth pressing or serpentine pressing etc
are being utilized for the separation of juice from berries. Juice obtained by
the conventional processing techniques reported to be turbid, with a high
content of suspended solids and pulp oil (177). Juice with pulp oil leads to the
formation of an undesirable oily layer on the top during storage.
Centrifugation of unheated juice causes rapid separation into a floating cream
Introduction
59
phase, an opalescent clear juice in the middle and sediment. Zhang et al;
suggested the use of a stalk centrifuge or a cream separator to separate the
cream from the juice (178). Beaveredge et al; suggested the removal of fat
from the centrifuged juice with minimum contamination to adjacent layers by
keeping at 4o C or lower temperature (147). Liu and Liu reported the use of
pectin methyl esterase to break down pectins in the pulp to obtain clear juice
(179). Heilscher and Lorber reported the use of crystalline sugar for
sedimentation and subsequent centrifugation for clear juice (180). Solvent
extraction has been tried for oil recovery, but it is not recommended for
nutraceutical applications owing to the residual solvents and the destruction of
bioactive phytochemicals during desolventisation. Fresh pressed juice
separates into three phases when allowed to stand overnight in the
refrigerator: an upper cream phase, juice in the middle portion, and sediment
at the bottom. Enzymatic hydrolysis with commercial, broad spectrum
carbohydrate hydrolyzing enzyme preparations reduces the juice viscosity,
assists juice separation, and provides an opalescent juice (147).
SC-CO2 extraction has been suggested for superior quality, solvent-
free oils. A theoretical model of SC-CO2 extraction of organic oil from SB
seeds is constructed by Derevich et al; (181). However, the berries must be
dried before supercritical extraction, resulting in a loss of juice and
phytonutrients during drying.
1.4.4 Physiological effects of SB berries
A wide spectrum of physiological effects of SB berries and berry
products has been reported, including AO (128,144,182,183), radio-protective
(184), anti tumor (185,186) inhibition of LDL cholesterol oxidation and
platelet aggregation (187), anti-hypertensive (188), immunomodulation and
cytoprotective effects (189), protection from gastric ulcer (190), reduction of
atopic dermatitis (191,192), and wound healing (193).
Introduction
60
Gao et al; investigated the AO activity of SB fruits and its relationship
with maturity (158). The study demonstrates that capacity of phenolic and
ascorbate extracts to scavenge radicals decreased significantly with increased
maturation and the changes were strongly correlated with the content of total
phenolics and ascorbic acid. AO capacity of the lipophilic extract increases
significantly with maturation and corresponds to the increase in total
carotenoids. Eccleston et al; reported that SB juice was rich in AO and
moderately decreased the susceptibility of LDL to oxidation (144). Alcohol
and water extracts of various SB seeds are found to possess high levels of AO
and antibacterial activities and these activities are attributed to the high
phenolic content in SB seeds (128,194). SB seed oil is reported to exert
protection from oxidative damage caused by SO2 exposure in mice (195).
Johansson and coworkers showed that SB oil inhibited platelet
aggregation in humans (188). A similar inhibitory effect to aspirin on platelet
aggregation induced by collagen in mouse femoral artery was reported for a
total flavone extract from SB (196). This ability to prevent in vivo
thrombogenesis suggested that SB fruit consumption may help prevent
cardiac and cerebral thrombosis in humans.
In contrast to the in vitro studies Suomela and coworkers reported that
SB flavonols, ingested with oatmeal porridge, do not have a significant effect
on the levels of oxidized LDL, C-reactive protein, and homocysteine, on the
plasma AO potential, or on the paraoxonase activity in human (197). They
also showed that flavonols in oatmeal porridge were rapidly absorbed, and a
relatively small amount of SB oil added to the porridge seemed to increase the
bioavailability of flavonols considerably.
In another study Nersesyan and Muradyan shown that SB juice
protects mice against genotoxic action of the anticancer drug cisplatin (198).
Introduction
61
Geetha et al; found that concentrated (500 µg/mL) alcoholic extracts of fruit
and leaves of SB could inhibit chromium-induced free radical apoptosis and
DNA fragmentation and restored AO status to that of control cells in a
lymphocyte in vitro model system. The leaf extracts have a cytoprotective
effect against chromium induced cytotoxicity as well as immunomodulating
activity (188).
The preventive effect of SB extracts on liver fibrosis was also
demonstrated through a clinical study (199). SB proanthocyanidins reported
to play an important role in healing of acetic acid-induced gastric lesions in
mice possibly by the acceleration of the mucosal repair (200).
Introduction
62
1.5. Relevance and Objectives of the Present Study
Epidemiological surveys have provided positive correlation between
diets rich in fruits and vegetables and the delayed onset of degenerative
diseases and ageing. Vast diversity and better productivity of natural products
towards drugs and drug leads over de novo molecules coupled with
epidemiological results favoring the therapeutic efficiency of herbals develop
an increased interest in natural products among health scientists recently. This
demands detailed phytochemical and pharmacological investigations on
plants along with standardization and evidence based validation of herbal
products. This also requisites the development of economically viable and
industrially adaptable processing techniques for plant products suitable for
nutraceutical and pharmaceutical applications.
SB is grown in cold regions of Asia, Europe and North America.
World annual production of SB berries is approxiamtely 200,000 tons and is
being used as food and for nutraceutical and cosmaceutical applications.
Complex nitrogen fixing root system enables this plant as a optimum pioneer
plant for eroded areas. In India, SB is grown in the Trans-Himalayan cold
deserts such as Ladakh, Lahaul, and Spiti at altitudes 2500–4500 m. H.
rhamnoides, H. salicifolia, and H. tibetana are the predominant SB species in
India. Of these, H. rhamnoides is widely distributed in the Trans-Himalayan
region. H. salicifolia, and H. tibetana are respectively endogenous to Trans-
Himalayan region and Indian Himalayas. In India 30,000 hectors of land is
under SB and act as a source of food, medicine and cattle feed as well as
income for villagers in under developed SB growing areas.
Introduction
63
Detailed reports on the phytochemical compositions of SB berries are
rather limited. In India, SB growing areas are under extreme climatic stress
with wide temperature variations (-40 to +40), low precipitation, high sun
light and arid soil. Since the emergence of secondary metabolites in plants is
associated with their defense and survival mechanisms, geo-climatic
variations might reflect in their phytochemical composition. SB in Indian
Trans-Himalayas has not been investigated in this perspective. In this study
the commonly cultivated H. rhamnoides berries were investigated in detail for
their chemical composition. Analytical methods suitable for the quality
evaluation of berries and berry products were also standardized. Nutritional
quality of the berries belonging to major species of SB grown in India
namely; H. rhamnoides, H. salicifolia, and H. tibetana was compared using
modern analytical techniques as a part of this investigation.
Even though more than hundreds of industries, mostly from China and
Russia are engaged in the processing of SB berries for value added products,
detailed reports with processing parameters and chemical evaluation of
process streams are not available. Indian Himalayas hosts world’s second
largest area under SB, however, so far, no attempt has been made to utilize
SB berries grown in this region. SB berries are highly perishable and have to
be processed within hours of harvesting to obtain quality products. Most of
the available process involves organic solvents for oil extraction which is not
suitable for ecologically fragile SB growing areas in Himalayas. This
demands a green process (solvent free) with minimum technicalities
applicable for fresh berries and suitable for ecologically sensitive areas.
Therefore, development of a green process for the production of pulp oil and
oil free clear juice with maximum retention of bioactive phytochemicals was
attempted here. SC-CO2 extraction conditions for oil from the seeds obtained
as a byproduct of the developed process was also optimized.
Introduction
64
A wide spectrum of biological activities has been attributed to SB
berries. Apart from the reports on applications and physiological properties of
SB, reports describing the bioactivities of SB berries in relation with their
phytochemical compositions are limited. This study was aimed at the
evaluation of AO properties of SB berries and chemical profiling of active
fractions. Extracts of anatomical parts of berries were prepared and subjected
to in vitro AO capacity evaluation. Active extracts were further fractionated
and AO capacity was evaluated. The AO active fractions were subjected to
detailed chemical composition analysis.
SB growing under unique geo-climatic conditions of Trans-Himalayan
region of India has not been investigated in terms of the detailed
phytochemical profile and chemical characterization, AO properties and
process development for products for nutraceutical and cosmaceutical
applications. Objectives of the present study was therefore framed, from the
above prospective. Thus this study was undertaken with the following
objectives;
1. Detailed investigations on the chemical composition of H.
rhamnoides berries in Indian Himalayan region.
2. Development of analytical protocols for major bioactive
phytochemicals in SB berries.
3. Quality evaluation of SB berries belonging to major species of SB
in India
4. Development of a green integrated process for fresh SB berries for
nutraceutical and cosmaceutical applications.
5. Evaluation of process streams and products in terms of their yield,
efficiency and chemical compositions.
6. Evaluation of in vitro AO capacity of SB berries
7. Chemical profiling of active berry extracts.
Introduction
65
8. Evaluation of structure-activity relationship of major AO active
compounds in SB berries.
Results of the present investigation are summarized and discussed as
Chapter-3-Results and Discussion. Chapter-3 is divided into 4 sections.
Section 3.1 deals with the detailed chemical profiling of common SB (H.
rhamnoides) berries in Indian Trans-Himalayas. Berries were separated into
pulp, seed coat and kernel and analyzed in detail for their lipid profile,
vitamin C content, and organic acid and phenolic composition. Analytical
protocols for the profiling of major bioactive phytochemicals in SB berries
were also standardized. Section 3.2 discusses the quality evaluation of berries
belonging to major SB species found in India. The berries were compared in
terms of their lipid profile, vitamin C and phenolic contents. Development
of a green process for the integrated processing of fresh SB berries for high
quality pulp oil, juice and seed oil is discussed in Section 3.3. The process
parameters and chemical composition of process streams and products were
evaluated. Section 3.4 deals with the AO capacity evaluation of H.
rhamnoides berries and chemical profiling of active fractions.
Summary and conclusion of the present study is included as Chapter 4
Introduction
66
Materials & Methods
67
Chapter 2
MATERIALS AND METHODS
Materials & Methods
68
I have not failed. I've just found 10,000 ways that won't work.
......Edison
Materials & Methods
69
2.1. Sea Buckthorn Berries
Berries of three SB species from major SB growing areas in the Trans-
Himalayan region of India were used in this study. Fresh berries of H.
rhamnoides var, turkestanica were collected from the Indus valley of Ladakh,
Spiti, and Lahaul regions. H. tibetana and H. salicifolia berries were collected
from Spiti and Lahaul regions, respectively. Berries were collected in
triplicate from three different locations (3 × 3) of the same geographic area
and labeled as Lahaul 1, Lahaul 2, and Lahaul 3. Berries were collected
during June to November of 2003 at the stage of commercial maturity, as
judged by juiciness and appearance. Berries were collected and identified
with the help of experts from the Field Research Laboratory (DRDO, India),
Leh, India and Himachal Pradesh Agricultural University, Kullu, India.
Berries were stored in polyethylene bags and ferried to the NIIST Trivandrum
by air under cold conditions and stored at −20°C until analysis.
Berries were separated into berry pulp, seed coat and kernel. Seeds
were separated from the berries just before analysis. Frozen berries were
taken from the freezer (−20°C), thawed at 25°C for 1 h, and crushed gently so
that the seeds remained intact and the seeds were then separated manually
from the pulp. Seeds were coarse powdered and seed coat was separated from
the kernel by winnowing.
2.2. Chemicals and Reagents.
Biochemical standards such as β-carotene, fatty acids, β-sitosterol,
stigmasterol, campesterol, quercetin, kaempherol, ascorbic acid, malic acid,
citric acid, gallic acid, protocatechuic acid, parahydroxybenzoic acid, vanillic
acid, salicylic acid, cinnamic acid, p-coumaric acid, ferulic acid, caffeic acid,
XO and allopurinol were obtained from Sigma-Aldrich (St. Louis, MO,
USA). Tocopherols and tocotrienols were purchased from Calbiochem
(Merck Ltd., Mumbai, India). Folin-Ciocalteau’s (F-C) reagent, and HPLC
Materials & Methods
70
grade hexane, isopropyl alcohol, methanol, water, acetic acid and formic acid
were obtained from Merck Ltd (Mumbai, India). Other chemicals and
reagents were of analytical grade. Isorhamnetin and quinic acid used were
isolated from SB berries and characterized by spectroscopic studies.
2.3. Proximate Composition of Berries.
AOAC methods were used for the determination of moisture (No.
930.15), protein (No. 988.01) and ash content (No. 942.02) (201). Moisture
was determined by oven dry method. Total fat and sugar were respectively
estimated by soxhlet and anthrone method. Analysis was carried out in
triplicate and mean value and standard deviation were determined.
Total protein: Most of the nitrogen in the plants occurs as proteins and
amino acids. Total amount of nitrogen in plant material is therefore indicating
the amount of total proteins and amino acids. Nitrogen content was estimated
by the Kjeldahl method. About 1g sample and 0.5g of digestion mixture (2.5g
SeO2 + 100g K2SO4 + 20g CuSO4) were weighed into a Kjeldahl’s flask. 10
mL conc. H2S04 was added and heated until color of the solution was changed
to clear blue green. This clear solution was made up to 100 mL under cold
conditions. The Kjeldahl apparatus was set up for protein estimation with 20
ml boric acid and 1 mL mixed indicator (bromocresol green + methyl red). 5
mL of digested sample with 20 ml 40% NaOH and 10 ml water were taken in
the apparatus connected to the steam line. Steam was bubbled through the
mixture and the NH3 evolved was stripped with steam and trapped in boric
acid. Upon ammonia evolution, color of boric acid changes to blue and the
steam bubbling is continued for 20 minutes. The resulted boric acid was
titrated with 0.1N HCl till blue color of the solution disappears. Amount of
nitrogen in the samples was calculated by the following relation
% of nitrogen = 14 x100 x N Hcl x 100
w x 5 x 1000
Materials & Methods
71
Where, ‘N Hcl’ and ‘w’ represents the normality of HCl used and weight of the
sample respectively. The amount of protein was expressed in terms of
glycine.
Total sugars – Anthrone method: In this method the total carbohydrate
was estimated by the dehydration of carbohydrate to furfural and its
subsequent condensation reaction with anthrone (9,10–dihydro–9–
oxoanthracene) to yield a blue green colored product. 1 g of substance was
dried in air oven, defatted with hexane and refluxed with 80% ethanol for 6
hours. Filtered and made up to 25 mL. 0.2 to 1 mL of glucose solution (0.1
mg/mL) and 0.2 to 0.8 mL of sample solution were pipetted in test tubes and
made up to 1ml with distilled water. 4 mL of anthrone reagent (0.5 g anthrone
and 10 g thiourea in 1L of 66% v/v of H2SO4 and stored at 4 oC) was added to
the test tubes and warmed to 80 to 90 oC for 15’. Cooled under tap water and
absorbance was measured at 620 nm in uv–visible spectrophotometer (UV –
2001, Shimadzu, Japan). The amount of carbohydrates in the sample was
determined by plotting the calibration curve using authentic standard of
glucose.
2.4. Extraction of Lipids-Christies Method:
1 g of berries was homogenized in 10 mL methanol for 2 min in a
blender, then 20 mL chloroform was added and homogenization continued for
5 min more. The mixture was centrifuged at 1500 × g and filtered. Solid
residue was resuspended in chloroform methanol (2:1 vol/vol, 30 mL) for 5
min. The mixture was centrifuged and filtered, and the residue was washed
with a chloroform/methanol mixture (1:1, v/v, 30 mL). The filtrates and
washings were combined, and one-fourth of the total volume 0.88% aqueous
potassium chloride solution was added, shaken well, and allowed to settle.
The lower layer was separated off and washed with methanol/water (1:1 v/v).
The purified lipid layer was filtered and dried over anhydrous Na2SO4, the
Materials & Methods
72
solvent was removed in a rotary film evaporator, and the amount of lipids was
noted. Lipids were stored in chloroform at −20°C for further analysis (171).
2.5. GC Profiling of Fatty Acids
Fatty acid methyl esters (FAME) of berry lipids were prepared
according to the IUPAC method (202). FAME were analyzed using a
Hewlett-Packard 5890 series II model gas chromatograph (Avondale, PA)
equipped with a flame ionization detector. The column used was an HP-FFA
(cross-linked FFAP: 30 m × 0.5 mm × 1 µm; Hewlett-Packard, Avondale,
PA). Injector and detector port temperatures were 250 and 300°C,
respectively. The column temperature was maintained at 100°C for 1 min and
then increased to 180°C at 5°C/min and maintained at that temperature for 15
min. Nitrogen was used as the carrier gas at 20 mL/min. FAME components
in the samples were determined by comparing the retention time (TR) with
that of standard FAME run under the same conditions. FA composition was
expressed as weight percentage of the total FA.
2.6. Total Carotenoids
Aliquots of lipids in hexane (0.1g/mL) was added to 0.5 mL of 5 g/L
NaCl, vortexed for 30 s and centrifuged at 1500 × g for 10 min (Cooling
Centrifuge C-30, REMI, India). The supernatants were diluted with hexane
and the absorbance at 460 nm was measured. The amount of carotenoids was
calculated by plotting a calibration curve with authentic standard of β-
carotene (1–10 µg/mL) and expressed in terms of β-carotene (158). The mean
± standard deviation of three replications was recorded.
2.7. HPLC Profiling of Tocopherols/Tocotrienols
Lipids were dissolved in hexane (1 mg/mL) and filtered through 0.45
µM PTFE membrane. A Shimadzu HPLC binary system (LC- 10A) with LC-
10AD pumps, a Rheodyne injector fitted with a 20 µL sample loop, a SPD-
Materials & Methods
73
10A UV-vis detector, and a CR 7Ae data processor for data acquisition,
analysis, and display was used for the analysis. A Phenomenex NH2 column
(5 µm, 4.6 X 250 mm; Luna) was used as a stationary phase with a mobile
phase of n-hexane and isopropanol (96:4 v/v) at a flow rate of 1 mL/min. The
UV detector was set at 297 nm. The HPLC conditions were standardized
using individual standards and their various mixtures (203). The mean ±
standard deviation of three replications was calculated.
2.8. HPLC Profiling of Sterols
Sterols in the lipid samples were hydrolyzed and analyzed as their
aglycones. Approximately 1 g of sample was refluxed with 6 mL methanol
and 1 mL 50% KOH (wt/v) for 3 h in a water bath. The unsaponifiable matter
was extracted with petroleum ether and desolventised in a thin film
evaporator under reduced pressure below 50 0C. The residue was redissolved
in chloroform, made up to a known volume with methanol and analyzed in the
same HPLC binary system used for tocopherol analysis, using a reverse phase
C-18 Zorabax column (4.6 × 250 mm) (Rockland Technologies, Newport,
USA) with methanol/water (96.5:3.5 v/v) at 1.5 mL/min as mobile phase. The
UV detector was set at 206 nm and a calibration curve was plotted with
authentic sterol standards (1–5 µg/mL) (204). The mean ± standard deviation
of three replications was calculated.
2.9 HPLC Quantification of Vitamin C
1 g of sample was homogenized repeatedly with 0.01 mol/L
metaphosphoric acid (6 X 20 mL) and centrifuged at 2200 × g for 5 min
(Cooling Centrifuge C-30, REMI). The supernatant was filtered through a
0.45 µm filter and analyzed immediately. Juice samples were also treated with
metaphosphoric acid, centrifuged and filtered. A 20 µL sample was injected
into a Shimadzu LC-8A HPLC equipped with a Phenomenex C-18 ODS-2
column (5 µm, 250 mm × 4.60 mm; Luna) and a PDA detector (SPD-M 10A
Materials & Methods
74
VP) set at 246 nm, and 3.7 mmol/L phosphate buffer (pH 4) at a flow rate of
1mL/min was used as mobile phase. Authentic standards of ascorbic acid
(0.5–5 µg/mL) were used for optimizing the HPLC conditions. Results are
reported as the mean ± standard deviation of three independently extracted
samples (205).
2.10. Organic Acid Analysis
2.10.1 Isolation of quinic acid: 100g freeze dried and defatted berry pulp was
extracted with water (3 X 250 mL) and dried under reduced pressure below
65o C. 10 g of this water extract was subjected to silica column
chromatography (silica 230-300 mesh) with a gradient mixture of ethyl acetate
and methanol. White crystalline powder obtained from polar fractions was
identified as quinic acid by spectral techniques.
Quinic acid:
Colourless needles (methanol) with melting point 162-163°C;
UV λλλλmax: 217 nm;
1H NMR (300 MHz, d4-MeOH): 1.81 (m), 1.99 (m), 3.93 (m), 3.41 (dd, J = 3,
9 Hz), 4.06 (q, J = 3, 6 Hz), 1.95 (m), 2.05 (m);
13C NMR :(75 MHz, d4-MeOH) 177.6 (C-7), 75.5 (C-1), 74.7 (C-3), 69.8 (C-
4), 66.2 (C-5), 36.6 (C-6).
2.10.2. HPLC profiling of organic acids: 1 g of sample was homogenized
repeatedly with HPLC-grade water (6 X 25 mL), clarified by centrifugation at
2000 × g for 15 min and filtered through a 0.45 µm filter. The juice was
further diluted with water, centrifuged and filtered. HPLC analysis was
carried out isocratically using phosphoric acid solution at pH 2.4 as mobile
phase at a flow rate of 0.7mL/ min. A Shimadzu LC-8A HPLC equipped with
a Phenomenex C-18 ODS-2 column (5 µm, 250 × 4.6mm; Luna)
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75
thermostatted at 250 C (CTO-10 AC VP Column Oven), a 20 µL loop and a
PDA detector (SPD-M 10A VP) was used for the analysis. Authentic
standards of quinic acid (1–10 µg/mL), malic acid (1–10 µg/mL), citric acid
(1–10 µg/mL), maleic acid (0.5–10µg/mL) and fumaric acid (0.5–10µg/mL)
were used to plot the calibration curve. Peaks in the samples were identified
by comparing their TR and uv spectra with those of reference compounds and
were quantified at 215 nm. The mean ± standard deviation of three
replications was determined.
2.11. Total Phenolics
1g of the sample was extracted (5 X 20 mL) with 70% methanol,
filtered, desolventised and made up to 25 mL with methanol. Juice was
clarified by centrifugation and supernatant was used for the analysis. Aliquots
of the extracts (0.5 mL) were oxidized with 0.5 mL of F-C reagent and the
reaction was neutralized with the addition of 1 mL 20% Sodium carbonate
(Na2CO3). The mixture was incubated at room temperature for 90 min. and
the absorbance of the resulting solution (blue color) was measured
spectrophotometrically at 760 nm. Calibration plot was obtained with gallic
acid and total phenolic content was expressed as milligram gallic acid
equivalents (GAE) per gram of extract/sample (206).
2.12. Flavonoids
2.12.1. Extraction of flavonoids: 10 g of the parts of berries was
homogenized with ethanol (6 x 50 mL) in presence of 250 mg of ascorbic acid
under nitrogen atmosphere at room temperature. The juice was clarified by
centrifugation at 1500 × g and the supernatant was used for analysis.
2.12.2. Total flavonoids: The amount of total flavonols was estimated by the
aluminum chloride method suggested by Chang et al (207). Quercetin, which
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76
is having a moderate absorbance, was used as standard. 10 mg of quercetin
was dissolved in 80% ethanol and then diluted to 25, 50 and 100 µg/mL. The
diluted standard solutions (0.5 mL) were separately mixed with 1.5 mL of
95% ethanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1M potassium
acetate and 2.8 mL of distilled water. After incubation at room temperature
for 30 min, the absorbance of the reaction mixture was measured at 415 nm
with a Shimadzu UV-160A spectrophotometer (Kyoto, Japan). The amount of
10% aluminum chloride was substituted by the same amount of distilled water
in blank. Similarly, 0.5 mL of extracts were reacted with aluminum chloride
for determination of flavonoid content as described above
2.12.3. Isolation of isorhamnetin. Freeze dried seeds (500 g) were powdered
and defatted with n-hexane (1L) and homogenized with methanol (4 X 1 L) at
room temperature. The extracts were desolventised under reduced pressure at
≤ 50 oC, to methanol extract (140 g). 100g extract was refluxed with methanol
(500 mL), deionised water (200 mL) and HCl (80 mL) for 2.25 h at 85 oC.
Methanol was removed and 500 mL water was added. Then the mixture was
neutralized with NaHCO3 and extracted with ethyl acetate (6 X 100 mL) and
the solvent was dried under reduced pressure at ≤ 50o C, to give aglycone
mixture (10 g). Ethyl acetate extract (5 g) was subjected to liquid
chromatography on silica gel (230-400 mesh) eluting with gradient mixtures
of n-hexane, ethyl acetate and methanol. Fractions (50 ml) were collected and
pooled according to their similarity on TLC to give four fractions (A1-A4).
Further repeated purifications of sub fractions A2 on liquid chromatography
afforded 20 mg compound pure flavonoid. This was subjected to spectral
analysis and identified as isorhamnetin.
Isorhamnetin: yellow crystals; m.p. 205-206° C;
EI-MS 316 (M+, 82.5), 301 (M+-15, 6.6), 208 (0.42), 108 (10.8), 123 (17.5),
85 (10), 151 (13.7), 57 (33.3), 40 (100);
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77
UV λλλλmax (MeOH): 254, 368; (+NaOMe) 268, 326, 414; (+AlCl3) 262, 425;
(+AlCl3/HCl) 260, 423; (+NaOAc) 273, 322, 387; (+NaOAc/H3BO3) 256,
363, 428 nm;
1H NMR (300 MHz, d5-py): 8.31 (1H, d, J = 1.7Hz, H-2’), 8.21 (1H, dd,
J = 1.9, 8.4 Hz, H-6’), 7.37 (1H, d, J = 8.5 Hz, H-5’), 6.87 (1H,d, J=1.8 Hz,
H-8), 6.76 (1H,d, J = 1.8 Hz, H-6), 3.84 (3H, s, OCH3).
Isorhamnetin was recrystalised from ethyl acetate-methanol mixture and used
as reference for the optimization of HPLC analytical conditions.
2.12.4. HPLC analysis of flavonoids: Flavonoids in the samples were
extracted with cold ethanol. The juice was clarified by centrifugation at 1500
× g and the supernatant was used for analysis. The berry extract and juice
were hydrolysed to obtain corresponding aglycones following the method
described by Stricher (208). About 20 mL of the juice or 1g of extracts was
refluxed with methanol (25 mL), deionised water (10 mL) and HCl (4 mL) for
2.25 h at 850 C. The solution was filtered, concentrated under reduced
pressure, made up to a known volume with methanol and stored at 40 C until
analysed. This homogenate was subsequently filtered through a 0.45 µm
(PTFE) filter and analysed in a Shimadzu LC-8A HPLC (Shimadzu
Corporation, Kyoto, Japan) fitted with a Rheodyne 7725 injector with a 20 µL
sample loop and a reverse phase C-18 ODS-2 column (Phenomenex,
Torrance, CA, USA) (5 µm, 250 mm × 4.6 mm; Luna) using methanol and
0.5% phosphoric acid (50:50 v/v) as mobile phase (1.5 mL/min).
2.12.5. HPLC-DAD-MS/MS analysis of flavonoid glycosides: HPLC-DAD-
MS/MS analytical conditions for the flavonoid glycoside profiling were
optimized. MS/MS conditions were optimized using rutin. Quantification of
the flavonoid glycosides were performed in DAD using the calibration plot
drawn with quercetin, kaempherol and isorhamnetin.
Materials & Methods
78
Instrument : Agilent 1200 HPLC with 6310 ion trap
Column: C-18, ODS-2, (5µ, 250X4.6 mm, Phenomenex, Luna)
Mobile Phase: Gradient of water, methanol and 10% acetic acid
Flow rate 0.8 mL/min
Time
(min.)
A (%,v/v)
(Water)
B (%,v/v)
(Methanol)
C (%,v/v)
(10% Acetic acid)
0 98 0 2
8 98 0 2
15 83 15 2
60 63 35 2
70 48 50 2
80 0 98 2
90 0 98 2
MS/MS Conditions
Capillary voltage 2500 V
Corona current 1000 nA
Nebuliser pressure 50 psi
Dry gas 5L/min
Drying temperature 350 oC
Vaporization temperature 250 oC
Ionization mode APCI+ESI
Polarity -ve
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79
2.13. Standardization of HPLC-DAD Analytical Method for
Phenolic Acids
2.13.1 Preparation of extracts: Five grams of the powdered berry parts were
homogenized at 200 rpm with methanol-water (60% for SB pulp and seed
coat, and 70% for kernel) mixtures (6 X 25 mL) at room temperature for 30
min. in nitrogen atmosphere. The mixture was centrifuged at 2000 X g for 15
min and supernatants were collected, combined and dried under vacuum at
≤50 ◦C and kept at −20 ◦C until analyzed.
2.13.2. Fractionation of phenolic acids: Free and bound phenolic acids in
the extracts were fractionated according to the procedure described by
Kozłowska et al and Zadernowski et al (209, 170). Crude phenolic extracts
were dissolved in 25 mL of water, acidified with 6N HCl to pH 2 and filtered
to remove the precipitated phospholipids. It was then extracted with diethyl
ether (5 X 25 mL) at room temperature. The diethyl ether extracts were
combined and evaporated under vacuum at ≤40o C and referred as free
phenolic acid fraction. The water phase was neutralized with 2M NaOH and
evaporated under vacuum at ≤50 oC almost to dryness. The residue was
treated with 20 mL 4N NaOH in nitrogen atmosphere for 4 h at room
temperature. The reaction mixture was then acidified to pH 2 with 6N HCl
and extracted with diethyl ether as before and analyzed as phenolic acids
liberated from esters. The water phase was then neutralized and dried as
before. The residue was hydrolyzed with 50 mL 2MHCl for 30 min at 95 oC.
The mixture was cooled, adjusted to pH 2 and extracted with diethyl ether as
before. This extract was referred to as the phenolic acids liberated from their
glycosides. The residual fatty material present in phenolic acid fractions thus
obtained were removed by dissolving in 20 mL 5% NaHCO3 solution and
extracting with diethyl ether (5 X 20 mL). Then the aqueous phase was
acidified with 6N HCl to pH 2 and extracted with diethyl ether as before. The
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80
extracts were dried and redissolved in 5mL methanol and kept at −20 oC till
analyzed.
2.13.3. Preparation of standard solutions: Standard solutions of nine
phenolic acids at a concentration of 0.5 mg/mL in methanol were prepared
and several dilutions in mobile phase were made. Solutions were filtered
through 0.45 µm PTFE filters, and injected directly.
2.13.4. Chromatographic conditions : A Shimadzu LC-8A HPLC system
(Shimadzu, Japan) with a binary solvent delivery system (LC-8A), a column
temperature controller (CTO-10AV), a Rheodyne injector with 20 µL sample
loop and a DAD detector (SPD-M 10 A) coupled with Class VP analytical
software were used for analysis. A reverse phase (RP) column (Phenomenex
C-18, ODS-2, 5µm, 250 X 4.6 mm) with an extended guard column was used
as stationary phase and column temperature was maintained at 35 oC. The
mobile phase was a gradient elution of water containing 2% acetic acid
(solvent A) and methanol (solvent B) at a flow rate of 1 mL/min.
The gradient program of mobile phase was as follows.
Time
(min.)
A (%, v/v)
(2% acetic acid)
B (%, v/v)
(Methanol)
0 100 0
5 100 0
10 90 10
15 90 10
20 80 20
25 80 20
25 40 60
30 0 100
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81
2.14 Analysis of PAs
2.14.1. Total PAs - Butanol–HCl Assay: 0.25mL of each extract (1 mg/mL)
was added to 3 mL of a 95% solution of n-butanol/HCl (95:5 v/v) in a
stoppered test tube, followed by 0.1mL of a solution of NH4Fe(SO4)2·12H2O
in 2M HCl. The tubes were incubated for 40 min at 95 oC. The absorbance of
the red coloration was read at 550 nm. Results were expressed in terms of
cyanidin chloride equivalents, as percentage of PAs on dry wt of the extract
(210).
2.14.2. Total PAs-Vanillin –HCl Assay: The content of PAs in extracts and
fractions was determined using modified vanillin assay and the results were
expressed as catechin equivalents (211). To 1 mL of catechin solution (0-300
µg/mL) or sample solution (200-300 µg/mL). 2.5 mL methanol (control) or
1% vanillin in methanol and 2.5 mL 9 mol/L HCl in methanol were added.
The reaction mixture was incubated for 20’ at room temperature and
absorbance at 500 nm was measured. The amount of PA in samples was
estimated from the calibration curve plotted with reference catechin (211).
2.14.3. Extraction of PA. Pulp, kernel, and seed coat were finally powdered
to an average particle size of 0.5 mm. Powdered samples (2 g) were extracted
with 20 mL methanol in water (100, 90, 80, 70, 60, 50, 25 & 0%) and acetone
in water (100, 90, 80.70, 60, 50, 25, & 0%). Similar methanol- water and
acetone -water mixtures containing 1% acetic acid were also used as
extraction solvents. For the extraction, the samples were homogenized at 200
rpm with solvent mixtures for 10’ and subsequently centrifuged at 2000 X g
(C-30, Remi, Mumbai India) and supernatants were collected. The residues
were resuspended in their respective solvent mixtures and the procedure was
repeated 2 more times. The supernatants were combined and dried under
reduced pressure at 40o C and redissolved in 25 mL methanol. The amounts of
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82
PA in these extracts were estimated by vanillin-HCl assay (211) and
expressed as catechin equivalents.
The solvent mixtures, as optimized in the previous step were used as
the extraction media for the preparation of crude phenolic extracts of SB
berries and leaves for further studies. 20 g of the powdered samples of SB
pulp, seed coat and kernel were extracted (5 X 100 mL), desolventised,
dissolved in methanol and stored at -20o C. Total phenolics and PA in these
extracts were estimated using F-C reagent (203) and vanillin-HCl assay (207)
respectively.
2.14.4. Purification of PA extracts. The crude phenolic extracts of SB
berries were purified with sephadex LH-20 column chromatography. Samples
(500 mg) of crude extracts were suspended in 95% (v/v) ethanol and applied
on to chromatographic column packed with sephadex LH-20 (2.3 X 40 cm)
and equilibrated with 95 % ethanol (v/v). The column was exhaustively
washed with 95% ethanol (v/v) and eluted with 25% acetone. Acetone elutes
were combined and dried under vacuum at 40 oC. The PA fractions thus
obtained were lyophilized and kept under refrigeration.
2.14.5. Average degree of polymerization (ADP). The ADP was estimated
using the purified PA fractions as the ratio of absorbance at 510 nm obtained
from butanol/ HCl reaction to the absorbance at 500 nm obtained from the
reaction with vanillin in acetic acid (212). Butanol/HCl assay was performed
as discussed in section 2.14.1. For vanillin assay vanillin reagent was
prepared by dissolving 0.5% vanillin in glacial acetic acid containing 4%
HCl. Extracts were dissolved in minimum quantity of methanol and diluted
with acetic acid. Incubated for 5’ and absorbance at 500 nm was measured.
ADP was calculated as the ratio of absorbance of butanol/HCl and vanillin
assay mixtures for equal quantities of samples.
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83
2.14.6. Depolymerisation of PAs in presence of phloroglucinol: A solution
of 0.1N HCl in MeOH, containing 50g/L phloroglucinol and 10g/L ascorbic
acid was prepared. 1 mL of purified PA extract in methanol (5g/L) was
treated with 10 mL of this solution for 20 min. at 50 oC and then combined
with 5 volumes of 40 mM aqueous sodium acetate to stop the reaction.
2.14.7. HPLC-DAD-MS/MS analysis of depolymerisation reaction
products.
Instrument : Agilent 1200 HPLC with 6310 ion trap
HPLC conditions:
Column: C-18, ODS-2, (5µ, 250X4.6 mm, Phenomenex, Luna) Mobile Phase-Gradient of 1% acetic acid in water Flow rate : 1 mL/min Time
(min.)
A (%,v/v)
(1% Acetic acid)
B (%,v/v)
(Methanol)
0 95 5 10 95 5
20 80 20 25 60 40 35 10 90 Calibration curves were plotted with catechin and epicatechin and the reaction products were quantified in terms of catechin and epicatechin.
MS/MS Conditions
MS ionization conditions were optimized by using catechin. Capillary voltage 2500 V Corona current 1000 nA Nebuliser pressure 50 psi Dry gas 5L/min Drying temperature 350 oC Vaporization temperature 250 oC Ionization mode ESI Polarity -ve
Materials & Methods
84
2.15. Processing of Fresh SB berries.
A schematic diagram of the patented method for processing fresh SB
berries is shown in Fig. 2.1. Batches of 90, 35 and 47 kg of fresh berries with
a moisture content of 70–75% were processed. Fresh SB berries taken from
cold storage were first cleaned by spraying with water to remove dirt and
other impurities and then blanched at 80o C to inactivate enzymes and to
facilitate the extraction of juice and oil in subsequent steps. The cleaned and
blanched berries were subsequently fed into a mechanical screw press of 100
kg/h capacity for dewatering. The compression ratio of the press was about 10
and the pressure was adjusted using the back-pressure control to separate
about 80% of the total water present in the fresh berries along with oil and
suspended solids. Fibrous press cake with unbroken seeds was obtained as a
residue. To achieve higher yields, three stages of extraction were performed,
the first with fresh berries and the second and third with the addition of 25%
by weight of warm water (60o C) to the residue. The juice obtained from all
three pressings was pooled and subjected to clarification. The pooled juice
was heated to 80o C with stirring in a stainless steel steam-jacketed kettle for
60 min. During clarification the pulp oil of orange/red colour was found to
float on the surface, while the suspended solids settled at the bottom. The
clarified juice was filtered while hot by passing it through a muslin cloth to
separate the debris and solids as sludge. The sludge was again mixed with hot
water (1:2) and the liquid phase containing residual oil was added to the
previous juice. Thus the liquid fraction was separated into juice with pulp oil
and solid sludge. The juice with pulp oil was then centrifuged (5000 × g, 60o
C) using a continuous centrifuge (RTA 1-01-025,GEA Westfalia Separator,
Oelde, Germany) to separate the fractions (clear juice, orange/red pulp oil and
residual solids as sludge). The fibrous cake with seed was dried in a crossflow
drier and separated into fibrous residue and seeds manually.
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85
Figure 2.1: Processing of fresh sea buckthorn berries.
Materials & Methods
86
2.16. SC-CO2 extraction of seed oil
SB seeds separated from the dried residue were ground in a laboratory grinder
(LB-105S, REMI, Mumbai, India) to an average particle size of 0.3 mm. The
ground seeds were extracted using a pilot-model supercritical fluid extraction
unit (SFE-2L, Thar Designs, Inc., Pittsburgh, PA, USA) at a temperature of
60o C, a pressure of 4.5 × 107 Pa and a gas flow rate of 60 g/min for 3 h. The
seed oil was collected in the cyclone separator of the extraction unit.
2.17. Activity Guided Fractionation of SB berries.
Phase I: Freeze dried berries were thawed at room temperature for 20 min.
and manually separated into seeds and pulp. Seeds were coarse powdered and
seed coat was separated from the seed kernel by winnowing. Pulp, seed kernel
and seed coat were finally powdered to an average particle size of 0.5 mm.
Powdered samples (100 g) were independently extracted with 1L (5 X 200
mL) hexane, ethyl acetate, methanol and water. For extraction, the samples
were homogenized at 200 rpm with solvents for 30 min in nitrogen
atmosphere and subsequently centrifuged at 2000 X g and supernatants were
collected. The residues were resuspended in their respective solvents and the
procedure was repeated 5 more times. The supernatants were combined, dried
under reduced pressure at ≤ 50 oC, and redissolved in 50 mL absolute
methanol. Extraction scheme is given in Fig-2.2.
Phase II. Kernel methanol extract with highest in vitro AO activity was
further fractionated between hexane, ethyl acetate, butanol and water
according to the scheme given in Fig -2.2.
5 g kernel methanol extract was made free from methanol, and
resuspended in 100 mL water. Then it was partitioned between the solvents (5
X 100 mL) in their increasing polarity order hexane, ethyl acetate, butanol
Materials & Methods
87
and water. Fractions thus obtained were desolventised under reduced pressure
and dissolved in 25 mL methanol. Yield and proximate composition of
fractions were noted and each fraction was evaluated for their in-vitro AO
activities. Ethyl acetate and water fractions of kernel methanol extract were
found to have high activity.
Phase III. Water fraction was further fractionated using sephadex gel LH-20
column chromatography. A dual pump flash chromatographic system
(BUCHI C-615.) equipped with a chromatographic column packed with
sephadex LH-20 (2.3 X 40 cm) at a flow rate of 10 mL/min was used for the
fractionation. 500 mg of water fraction was suspended in 95% (v/v) ethanol
and applied to the column and equilibrated with 95 % ethanol (v/v). The
column was washed with 95% ethanol (v/v) for 30 min and eluent was
collected as fraction-1. Then a gradient of acetone (Pump A) and water (Pump
B) was used for elution (Table 2.1). 100, 90, 80, 70, 60, 50 and 25% of
acetone were used for the elution, each solvent mixture was run for 30 min
and fractions 2 to 8 were collected. Fractions were dried under vacuum at 40o -
C, lyophilized and kept under refrigeration. Schematic presentation of the
fractionation is given in Fig-2.2
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88
Figure 2.2: Activity guided extraction scheme of SB berries
water
Hexane Butanol
Water
Ethyl acetate
Phase III
LC-MS/MS profiling
Sephadex LH-20 Column fractions
Chemical profiling
SB Berries
Pulp Seed Kernel Seed Coat
Hexane Methanol Water Ethyl acetate
Phase I
Phase II SB kernel Methanol
Extract
F-1 F-8
8
F-6
F-5
F-4
F-3
F-7
F-2
2
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89
2.18. In vitro AO Capacity Assays.
2.18.1. DPPH radical scavenging capacity: DPPH radical scavenging
activity of SB samples and reference compounds were measured according to
the method reported by Singh, et al; with slight modifications (213). Assay
was performed in 3mL reaction mixture containing 2.9 mL of 0.1 mM DPPH
solution in methanol and 0.1 mL of methanol (as control) or sample in
methanol (as test). The mixture was shaken vigorously and left to stand for 20
minutes at room temperature in the dark. The optical density (OD) of the
resulting solution was measured spectrophotometrically at 517 nm. The
capability to scavenge the DPPH radical was calculated using the following
formula:
Scavenging Activity (%) = [1- (OD of sample /OD of control)] x 100
2.18.2. ABTS radical scavenging capacity: ABTS radical cation scavenging
assay was carried out by the method of Re et al (214). ABTS radical cation
was generated by adding 7 mM ABTS to 2.4 mM potassium persulphate and
the mixture was allowed to stand for overnight in the dark at room
temperature. This solution was diluted to obtain an absorbance of 0.7 ± 0.05
with ethanol at 734 nm (Schimadzu UV-Vis spectrophotometer model 2450)
and was added to various concentrations of samples and reference compounds
(2-100 µg). The solution was shaken thoroughly and the absorbance was
measured after 7 min. The capacity to scavenge the ABTS radical cation was
calculated using the formula,
ABTS radical cation scavenging capacity (%) = [(A1-A2)/ A1] X100
where A1 is the absorbance of ABTS solution without test sample and A2 is
the absorbance of ABTS solution with test sample. Trolox was used as
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90
reference compound and the ABTS radical scavenging activity was expressed
as trolox equivalent antioxidant capacity (TEAC).
2.18.3. Fe(III) reducing power: The reducing powers of SB samples and
reference compounds were determined according to the method reported by
Yen and Chen (215). Different concentrations of samples/standards were
added to 2.5 mL of sodium phosphate buffer (0.2 M, pH 6.6) followed by the
addition of 2.5 mL of 1% potassium ferricyanide [K3Fe(CN)6]. The reaction
mixture was incubated at 50o C for 30 min. at the end of which 2.5 mL of
10% trichloroacetic acid were added, and the mixture was centrifuged at 5000
rpm for 10 min. The supernatant (2.5 mL) was mixed with distilled water (2.5
mL) and FeCl3 (0.1%, 0.5 mL), and the OD was measured at 700 nm using
spectrophotometer (Shimadzu-2450). Increased absorbance of the reaction
mixture indicated increased reducing power.
2.18.4. Fe(II) chelation capacity: The Fe(II) chelating potential of SB
samples and reference ccompounds was investigated according to the method
of Dinis et al with certain modifications (216). Briefly the reaction mixture
containing different concentrations of samples/standards (50 - 2500µg/mL),
0.25 mL FeCl2 (1mM) and 1mL ferrozine (5mM) was adjusted to a total
volume of 2mL with methanol, shaken well and incubated for 10 minutes at
room temperature. The absorbance of the mixture was measured at 562 nm
against blank. EDTA was used as a positive control. Percentage of iron
chelation was calculated using the formula;
Chelation effect (%) = [1- (Absorbance of Sample /absorbance of Control)] x
100.
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91
2.19.5. Hydroxyl radical scavenging activity. 0.1 mL EDTA (1 mM), 0.01
mL FeCl3 (10mM), 0.1 mL H2O2 (10 mM), 0.36 mL of deoxyribose (10
mM), 1 mL of extract concentrations (10-100 µg/mL), 0.33 mL phosphate
buffer (50 mM, pH=7.4) and 0.1 mL of ascorbic acid (1 mM) were added in
sequence. The mixture was incubated at 37o C for 1 hr. 1 mL of the incubated
mixture was mixed with 1 mL of 10% trichloroacetic acid and 1 mL of
thiobarbituric acid, 0.025 M NaOH and heated for 1 hr on water bath at 800 C
and a pink chromogen developed, which was measured at 523 nm (217).
2.17.3. Superoxide radical ion scavenging (SOS) activity: The superoxide
radicals were generated. The scavenging activities of SB samples and
reference compounds were determined by xanthine-XO system coupled with
nitro-blue tetrazolium (NBT) reduction (218). In this method superoxide
radical reduces the yellow dye (NBT2+) to produce the blue formazan, which
is measured spectrophotometrically at 560 nm. The capacity of the extracts
and standard compounds to scavenge superoxide radical was assayed as
follows: the reaction mixture contained 50 µl of 10 mM xanthine in 0.1 N
NaOH, 790 µL of Tris buffer (pH 7.4) containing 0.1 mM EDTA, 20 µL of
2.5 mM NBT in Tris buffer, and 0.1 mL extract or standard compound (as
test) or 0.1 mL tris buffer (as control). The reaction was initiated by the
addition of 40 µL of XO (0.04 U/ml) in Tris buffer (pH 7.4) and incubated at
25 oC for 20 minutes. Optical density of the resulting solution was measured
at 560 nm using spectrophotometer (Shimadzu-2450). The ability to scavenge
superoxide radicals was calculated using the following equation:
Scavenging Activity (%) = [1- (OD of Sample /OD of Control)] x 100.
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92
2.19.4. XO inhibitory activity: XO inhibitory activity was measured
spectrophotometrically by measuring uric acid formation at 290nm with
xanthine as substrate (219). The assay system consisted of 1mL reaction
mixture containing 0.1U/mL XO with 0.2 mL xanthine (0.26 µM) in 50 mM
phosphate buffer (pH=7.4). The reaction was initiated by adding the enzyme
with or without inhibitors and the change in absorbance of the mixture at 290
nm for 10’ was measured against a blank prepared in the same way but
replacing XO with phosphate buffer. Another reaction mixture was prepared
(control) without test compounds in order to have a maximum uric acid
formation. Various concentrations of allopurinol were used as a positive
control. The substrate concentration was kept less than 1µM to avoid the
substrate inhibition. The % inhibition of uric acid formation was calculated by
comparison with control and expressed as
XO inhibition (%) = [(A1-A2)/ A1] X100,
where A1 is the change in absorbance per minute without the sample and A2 is
the change in absorbance per minute with the sample.
2.20. Statistical analysis
SD and ANOVA calculations were performed using Microsoft Office
Excel 2003. Graphical calculations were performed using Microcal Origin 6.0
Professional.
Results & Discussion
93
Chapter 3.0
RESULTS AND DISCUSSION
Results & Discussion
94
If I have seen further (than certain other man) it is by standing upon the shoulders of giants
......Newton
Results & Discussion
95
3.1. Phytochemical Profiling of H rhamnoides
Berries of Trans-Himalayan Origin
Phytochemical profile of plants largely depends on the agricultural
practices and geo-climatic conditions. This demands detailed profiling of each
plant with respect to their genetic variations and geo-climatic area of
cultivation. Sea buckthorn (SB) grows in cold regions of Asia, Europe, and
North America. Indian Himalayas host world’s second or third largest area
under SB (30,000 – 40,000 ha). In India it grows in the cold and arid regions
of Trans-Himalayas at an altitude from 2500 m to 4500 m. In Ladakh it grows
approximately 11,500 hector area and covers major part of the forest area in
the region. It has been found growing abundantly in such adverse conditions
where temperature varies between -30 to 30o C and having annual precipitation
only 9 cm. It also grows wild in cold deserts such as Lahaul and Spiti in
Chamba and Kinnaur districts of Himachal Pradesh. SB in Trans-Himalayan
region of India is represented by three species viz; H. rhamnoides var.
turkestanica, H. salcifolia and H tibetana. Among these H. rhamnoides var.
Turkestanica is the most common in India. The SB-growing areas in the
Trans-Himalaya host unique geo-climatic conditions of high altitude coupled
with extreme temperature variations (–30 to 30°C), low precipitation, and low
oxygen in air. In these cold deserts plants are under extreme climatic stress and
are expected to have a distinct phytochemical profile. There were no
systematic studies to record the phytochemical profile of SB berries of Trans-
Himalayan origin. In this chapter detailed chemical profiling of SB berries
belonging to the most common species in India i.e. H. rhamnoides from
Ladakh region was attempted.
H. rhamnoides berries from Ladakh region was orange red in color. The
oval shaped berry had a hard seed enveloped with juicy pulp. The berries were
Results & Discussion
96
4-6 mm in diameter and 20-40 mg in weight. The berries were separated into
pulp, seed coat and kernel and analyzed for their chemical constituents in
detail. They were characterized in terms of their proximate composition, lipid
profile, phenolic and organic acids composition.
3.1.1. Proximate Composition:
Proximate composition of pulp and seed of H. rhamnoides berries was
determined following the standard methods and the results are presented in
Table 3.1. Pulp and seed showed considerable variation in terms of their
Figure 3.1: Anatomy of Sea Buckthorn berries
Results & Discussion
97
proximate composition. Fresh berry pulp had 81.3% moisture, 4.5% fatty
matter, 2.0% protein, 8.5% carbohydrate and 1.0% minerals. Seed contained
64.0% moisture, 7.2% fatty matter, 2.3% protein, 18.2% carbohydrate and
3.1% minerals. Fiber content in the berries was also estimated (Table 3.1).
Pulp and seed respectively had 1.2 and 0.7% fiber.
Reports on the proximate composition of H. rhamnoides berries of
Indian Trans-Himalayan origin are fragmented. Present study showed that
both pulp and seed of Indian H. rhamnoides berries are good source of
medicinal oil. The oil content obtained in the present study was in accordance
with the previous reports on H. rhamnoides berries of different origin. From
the previous reports it is clear that the oil contents in the pulp and thus in the
whole berries varied considerably with origins and other factors
(129,139,147,148,154,171,220). In fresh berries of H. rhamnoides, the oil
level ranges from 1.4% in sub species (ssp) sinensis from China up to 13.7%
in subsp. turkestanica from the Western Pamirs (154).Within a single
population, the oil content in the berries correlates with morphological
characteristics such as size and color of the berries, which may lead to a
difference up to 2-fold (129,154). In addition, harvesting time influences oil
content in the berries (129,148,154). The oil content in seeds of H.
rhamnoides is commonly ~10%, although higher values (up to 15–16%) have
been reported in some cultivars and wild berries from the Altai, Czech
Republic and Tajikistan (129, 139,148, 220,171).
Proximate analysis showed that H. rhamnoides berry was not a good
source of protein. Carbohydrate content in the seed was much higher than that
in pulp. Glucose, fructose, mannitol, sorbitol, xylose and xylitol are the main
sugars reported in H. rhamnoides berries of Chinese origin (127,147). There
are no reports on the detailed profiling of carbohydrates in the seeds
Results & Discussion
98
Table 3.1. Proximate composition of H. rhamnoides berry pulp and seed
(on fresh wt., Mean % ± SD)
Characteristics (%) Pulp seeds
Moisture 81.3 ± 1.6 64.0 ± 0.9
Fatty Matter 4.5 ± 0.4 7.2 ± 0.6
Protein 2.0 ± 0.2 2.3 ± 0.4
Sugar 8.5 ± 0.7 18.2 ± 1.1
Minerals 1.0 ± 0.2 3.1 ± 0.2
Crude Fiber 1.2 ± 0.3 0.7 ± 0.2
3.1.2. Fatty Acid Composition of Pulp and Seed Oils
Pulp and seed oils, extracted by Christies’ method were subjected to FA
composition analysis. Methyl esters of the FA (FAME) were prepared and
subsequently analyzed using GC. The individual FAs were identified by
comparing their retention time with that of reference FAME. The FA
compositions of pulp oil and seed oil are summarized in Table- 3.2. In the oil
from berry pulp, the dominating FAs were palmitoleic (48.2%), palmitic
(32.5%), and oleic (12.9%) acids. 3.5% linoleic, 0.5% stearic and 0.5%
linolenic acids were also obtained. FA profile of seed oil was different from
that of pulp oil. Seed oil contained linoleic (26.2%), linolenic (18.1%) palmitic
(17.7%) and oleic (12.9%) acid as major FA. Small amounts of palmitoleic
(3.5%) and stearic (3.1%) acids were also found in seed oil. While 16:1 acid
(48.2%) was the most predominant FA in pulp oil, its content in seed oil was
as low as 3.5%
Results & Discussion
99
Table 3.2. FA composition of pulp and seed oils of H. rhamnoides (wt % ±
SD)
C16:0 C 16:1 C 18:0 C18:1 C18:2 C18:3
Pulp oil 32.5 ± 1.4 48.2 ± 3.1 0.5 ± 0.9 12.9 ± 0.1 3.5 ± 0.6 0.5 ± 2.5
Seed oil 17.7 ± 1.2 3.5 ± 1.1 3.1 ± 0.2 29.5 ± 2.3 26.2 ± 2.2 18.1 ± 1.9
C16:0 - Palmitic acid C16:1 - Palmitoleic acid C18:0 - Stearic acid
C18:1 - Oleic acid C18:2 - Linoleic acid C18:3 - Linolenic acid
The FA profile obtained here for pulp oil is in accordance with the
previous reports on SB of Chinese and European origin. Palmitoleic (16–
54%), palmitic (17–47%) and oleic (2–35%) acids are reported as the
dominating FA in the soft parts of H. rhamnoides berries (139,145,148,220).
12.1 – 39.0% palmitoleic acid is reported in the pulp oil of berries of Chinese
and Finnish origin (129). Vereshchagin et al; reported that the Central Asian
and Baltic forms contained higher proportion of palmitoleic acid in the TAG
of berry pulp (55.0 and 42.0%, respectively) and Caucasian form had 16.0%
(150). The FA composition of the pulp oil of a Siberian form reported by
Ozerinina et al; is similar to that of the Central Asian and the Baltic form
(149). Cakir reported palmitoleic (47.9 %) and palmitic (25.0%) acids were
the major FAs in mesocarp oil of H. rhamnoides from Turkey (173). Genetic
factors (i.e. differences between species and subspecies) play a major role in
the variations of FA composition (148). In ripe berries, the FA composition in
the soft parts varies with harvesting time. In berries of same origin, the FA
compositions of seeds and soft parts remain rather constant among different
harvesting years (148). The variation in the FA composition, especially in the
soft parts of the berries, provides a great potential for industrial application
and plant breeding when seeking extreme sources of certain FAs.
Results & Discussion
100
The occurrance of palmitoleic acid in large proportion as shown in this
study is unique among vegetable oil as no other oil of plant origin is reported
to contain palmitoleic acid in substantial amount. Palmitoleic acid is reported
to facilitate cell membrane fluidity in a manner similar to that of PUFA but
with low susceptibility to oxidation. An increase in the level of dietary
palmitoleic acid has been suggested to improve the metabolism of vascular
smooth muscle cells (221). Hypocholesterolemic and hypoglyceridemic
activities comparable to that of linoleic and linolenic acids also have been
reported for palmitoleic acid (222).
Seed oil had oleic acid as the major FA (29.5%) followed by linoleic
acid (26.2%) and α-linolenic acid (ALA) (18.1%). The proportions of linoleic
acid and ALA reported in seed oil are in the range of 30–40 and 20–35%,
respectively. Other major FAs reported in seeds are oleic (13–30%), palmitic
(15–20%), stearic (2–5%), and vaccenic (18:1 n-7, 2–4%) acids
(139,145,220). Inspite of the differences in some morphological
characteristics and in growth conditions, subsp. sinensis (from China),
mongolica (from Russia) and rhamnoides (from Finland) have almost
identical FA composition in seeds (145, 148,154). Present study showed that
H rhamnoides seeds of Indian origin had slightly different FA profile with
oleic acid as the major FA followed by linoleic and ALA. ALA is ubiquitous
in plant leaf lipids and is present in some commodity seed oils: 8 to 10% in
soybean and canola, >50% in linseed oil, and 65 to 75% of perilla oil. The
seed oils of many Labiatae species have >50% ALA (222). Linoleic and
linolenic acids are considered as essential FAs. These FAs are having
significant role in neural and visual development and are precursors for
biologically active metabolites, the eicosanoids. As membrane components,
they have significant role in maintaining the fluidity and permeability of
biological membranes (223).
Results & Discussion
101
3.1.3. Carotenoids in Pulp and Seed Oils
More than 600 carotenoid compounds have been isolated and
characterized from natural sources. In plants carotenoids are involved in
photo protection or light collection. Most carotenoids contain an extended
array of double bonds which are responsible for their AO activity. Among the
carotenoids only α-carotene, β-carotene, γ-carotene and β-cryptoxanthine can
be converted into vitamin A. Nutritional significance of carotenoids in
prevention of cancer, heart disease, and age related macular degeneration
(AMD) has been suggested (224-226).
Total carotenoids in the pulp and seed oils was quantified and
expressed in terms of β-carotene, (Table-3.3). Pulp oil was found to have
significantly higher amount of carotenoids (2548 mg/Kg). Seed oil contained
moderate amount of carotenoids (389 mg/Kg). Berry pulp and seed
respectively contained 115 and 28 mg/kg (Fresh wt) of carotenoids
The carotenoid content in the berries are subjected to extreme
variation; differences up to 10-fold has been reported even within the same
natural population and subspecies. Total carotenoids (calculated as β-
carotene) from 1 to 120 mg/100 g in fresh berries have been reported (129).
Both raw material and methods of oil isolation influence the content of
carotenoids in the oils. Common levels of carotenoids are 1000–5000 mg/kg
in pulp oil and 200–1000 mg/kg in seed oil (129). The concentration in seeds
is typically 1/20–1/5 of that in soft parts (129). β-Carotene constitutes 15–
55% of total carotenoids, depending on the origin (129). α-carotene, γ-
carotene, dihydroxy-β-carotene, lycopene, zeaxanthin and canthaxanthin have
been reported to as the other carotenoids in SB berries (129).
Epidemiological studies suggest β-carotene as an important dietary
anticarcinogen as well as a protective factor against heart disease. β-Carotene
Results & Discussion
102
and other carotenoids are important AOs in plants. Carotenoids scavenge
singlet oxygen generated by the interaction of light with chlorophyll, before
the singlet O2 damages the plant cells. Carotenoids inhibit lipid peroxidation
and also quench many other free radicals. The free radical scavenging
activities of carotenoids can be attributed to the stability of carotenoid radicals
by the delocalization of unpaired electrons over the carotenoid conjugate
polyene system (227). Many studies have demonstrated that β-carotene
inhibits auto oxidation of lipids in biological tissues and food products.
Peroxyl radicals in particular, have been shown either to add to the long chain
of conjugated double bonds present in carotenoids, or to take part in electron
transfer reactions giving rise to carbon centered β-Carotenyl free radicals
(228):
β-Carotene + ROO● ----------------- [ROO- β-Carotene],
or [ROO▬ + β-Carotene
●]
Table. 3.3. Carotenoid and tocol content of pulp and seed oils of H
.rhamnoides berries (mg/kg of oil, Mean ± SD)
Carotenoids
(mg/kg)
Tocopherols (mg/kg).
ααααT1 αααα T3 ββββ T Γ T γ T3
+δδδδ T δδδδ T3 Total
Pulp
Oil
2548
± 29
954
±73
95
±15
27
±6
126
±5
187
±11
14
±2
1394
±87
Seed
Oil
389
± 17
608
±45
10
±1
21
±1
433
±37
496
±12 tr
1193
±51
•••• T1 = tocopherol, T3 = tocotrienol
Results & Discussion
103
3.1.4. Tocopherols and tocotrienols (tocols) in pulp and seed oils
Compounds showing vitamin E activities are termed as tocopherols. In
nature eight chemical constituents have been found to have vitamin E activity,
they are d-α, d-β, d-γ and d-δ tocopherols and d-α, d-β, d-γ and d-δ
tocotrienols (Fig 3.2). These are fat-soluble in nature and also act as major
radical scavengers in membranes. Vegetable oils such as wheat germ oil, palm
oil and rice bran oil provide the best source of these bioactive phytochemicals
essential in human nutrition.
Tocols in the pulp and seed oils of H rhamnoides were profiled in RP-
HPLC (Figure 3.3). Individual tocols in the samples were identified by
comparing their TR (retention time) with that of reference compounds. The
amount of tocols was calculated from the calibration curve plotted with
reference compounds. HPLC analysis showed that both pulp and seed oils
were significantly rich in tocols (Table-3.3). Pulp oil had 1394 mg/kg of tocols
and α-tocopherol (954 mg/kg) contributed 68.4% of total tocol. γ-tocopherol
(126 mg/kg) and α-tocotrienol (95 mg/kg) were the other major tocols in the
pulp oil. Under the chromatographic conditions used here, the resolution
between γ-tocotrienol and δ- tocopherol was poor, therefore they were
calculated together. γ-tocotrienol and δ- tocopherol together contributed 187
mg/kg. Presence of β-tocopherol (27 mg/kg) and δ- tocotrienol (14 mg/kg)
were also detected. 1193 mg/kg of tocols was obtained for seed oil. Seed oil
also had α-tocopherol (608 mg/kg) as the major tocol, which contributed 51%
of total tocols. In contrast to the pulp oil, seed oil had high amount of γ-
tocopherol (433 mg/kg). Presence of α-tocotrienol (10 mg/kg), β-tocopherol
(21 mg/kg) and δ- tocotrienol were also observed in seed oil. γ-tocotrienol and
δ- tocopherol together contributed 496 mg/kg.
Results & Discussion
104
This is the first detailed report on the tocol composition of SB berries of
Himalayan origin. Both pulp and seed oil had significantly higher amount of
tocols than those in common vegetable oils like rice bran (980 mg/kg), soya
bean (960 mg/kg), palm (1180 mg/kg), sesame (290 mg/kg ), sun flower (550
mg/kg) and safflower (800 mg/kg) oils (229). According to the available
reports on SB berries belonging to other regions the total tocol content vary
within the range 100–300 mg/kg in seeds and 10–150 mg/kg in fresh berries
(145,171,129). In the pulp of H rhamnoides berries of Chinese origin α-
tocopherol alone constitutes up to 90% of the total tocols, while both α-and γ-
isomers (each representing 30–50% of total) are the major ones in seeds. α, β,
and γ-tocotrienols each accounts for 0.5–5% of total tocopherols and
tocotrienols in soft parts, whereas in seeds the β isomer (2–8%) clearly
dominates accompanied by only trace amounts of α and γ-isomers
(145,171,129). The tocol profile obtained for the SB berries of Indian origin
are matching with the reports available on berries of Chinese and Polish
origin. A tocopherol profile of 75–89% of α-tocopherol, 4–11% of γ-
tocopherol, 2.4–12.2% of β-tocopherol, 0.3–2.4% of δ-tocopherol, 0.4–4.8%
of β-tocotrienol, 0.4–3.2% of α-tocotrienol, and 0.6–2.5% of δ-tocotrienol was
reported for the pulp of berries of the sinesis and mongolica subspecies of H.
rhamnoides from China (171). Zadernowski et al; reported 101–128 mg/100 g
of tocopherols in pulp oil of H. rhamnoides berries of Polish origin with a
profile of 62.5–67.9% of α-tocotrienol, traces of γ-tocotrienol, and 30–40% of
δ-tocopherol. The amount and composition of tocols in the berries largely
depend on the species, geo-climatic variations and maturity (157).
The beneficial effects of vitamin E compounds include AO activity,
lipid lowering effect, nutrient function, prevention of degenerative diseases
like CVD, cataract, arthritis and cancer, and immune system maintenance
(230,231). Quireshi et al; demonstrated that tocotrienols inhibited the activity
of 3-hydroxy-3-methyl glutaryl coenzyme A (HMGCoA) reductase, a rate
Results & Discussion
105
limiting enzyme in cholesterol synthesis (232). Tocopherols and tocotrienols
seem to be the most efficient lipid AOs in nature. Tocopherols inhibit lipid
peroxidation by scavenging lipid peroxyl radicals much faster than these
radicals can react with adjacent FA side chains or with membrane proteins.
The –OH group of the tocopherol gives up its hydrogen atom to the peroxyl
radical and quench it. This leaves a tocopheryl radical. The tocopheryl radical
is stabilized by the delocalization of unpaired electron over the conjugated
polyene or aromatic system. The tocopheryl radical is terminated by the
polymerization reaction or by some regenerating mechanism in the body.
Tocotrienols exerts greater AO than tocopherols (233).
Figure 3.2. Tocopherols and tocotrienols
R1 R2 R3 (A) Tocopherols (B) Tocotrienols
CH3 CH3 CH3 α α
CH3 H CH3 β β
H CH3 CH3 γ γ
H H CH3 δ δ
α-Tocopherol. α –Tocopherol Lipid-O2 Lipid –O2H
O
CH3 CH3
CH3
CH3
CH3
R1
R3
R2
HO
O
CH3 CH3
CH3
CH3
CH3
R
R
R
HO
(A) Tocopherols (B) Tocotrienols
Results & Discussion
106
Figure 3.3 HPLC profile of tocols in H rhamnoides pulp oil at 295 nm.
Minutes
0 5 10 15
Volts
0.00
0.02
0.04
3.1.5. Sterols in Pulp and Seed oils
In plants sterols are usually found as their glycosides. Steroidal
glycoside in pulp and seed oil were hydrolyzed to their aglycones and
subsequently analyzed in HPLC (Figure 3.5). Sterols in the samples were
identified by comparing the retention time with that of reference compounds.
Amounts of individual sterols were determined from the calibration curves and
are given in Table 3.4.
The β-sitosterol (3034 mg/kg) and stigma sterol (1062 mg/kg) were
found to be the major sterols in the pulp oil. In comparison to pulp oil, seed oil
was found to be a richer source of phytosterols with 16200 mg/kg of sterols. β-
sitosterol was found to be the major sterol along with stigmasterol and
campesterol in SB seed (Figure 3.4).
1
2
5
4
3
1: αααα-Tocopherol, 2: αααα-Tocotrienol, 3:ββββ-Tocopherol,4: γ- Tocopherol,
5: γ- Tocopherol + δδδδ- Tocopherol
Results & Discussion
107
According to the previous reports the sterol content in SB berries is in
the range of 0.02–0.04% in the soft parts and 0.1–0.2% in seeds (both based
on fresh weight) (154). In fresh berries of subsp. sinensis and rhamnoides the
sterol content reported to fall in the range of 350–500 mg/kg, of which 70–
80% is in the soft parts (154). Typical values for sterol content are 1–2% in
seed oil and 1–3% in oil from the soft parts (154,234). Sitosterol constitutes
60–70% of seed sterols and up to 80% of those in soft parts. Isofucosterol is
another major sterol representing 10–20% of seed sterols and 2 –5% of sterols
of the soft parts. Campesterol, stigmastanol, citrostadienol, avenasterol,
cycloartenol, 24-methylenecycloartanol, obtusifoliol, each represents 1–5% of
total sterols of both fractions (154,234). The amounts of identified sterols in
pulp and seed oils obtained in the present study are in accordance with the
previous reports. Some minor peaks in HPLC profiles of both pulp and seed
oils could be identified as sterols, which might have further contributed to the
total sterol content.
Table 3.4. Sterols in H. rhamnoides pulp and seed oils (mg/kg ± SD )
Sterols Pulp Oil Seed Oil
β-sitosterol 3034 ± 84 13305 ± 132
Stigmasterol 1062 ± 67 1125 ± 111
Campesterol Tr 1770 ± 50
Total 4096 ± 98 16200 ± 260
Results & Discussion
108
Figure 3.4 HPLC profile of sterols in H rhamnoides seed oil.
CH3
HO
CH3
CH3
CH3
CH3CH3
ß sito sterol
CH3
HO
Campesterol
CH3
CH3
CH3
H3C
H3C
CH3
CH3
HO
CH3
CH3
CH3
Stigma sterol
Figure 3.5 Structures of major sterols in SB berries.
SS: stigmasterol, CS: campesterol, BS: β-sitosrerol
Results & Discussion
109
3.1.6. Vitamin C Content in the Berries:
Like tocopherol, vitamin C (ascorbic acid) has been defined as an
essential nutrient. Vitamin C is common in a variety of fruits and vegetables.
Being a biologically essential phytochemical for human, vitamin C content in
H. rhamnoides berries was analyzed. It was revealed that soft parts (fresh) of
rhamnoides berries contained significantly higher amount of vitamin C (2232
mg/kg) (Table 3.5). Several authors have reported the high vitamin C content
in Chinese berries (3600-25000mg/kg) (127), however the reports about the
nutritional qualities of Indian berries are rather limited. SB berries are well
known for its extraordinarily high levels of vitamin C, and therefore
considered as an important quality parameter of the SB juice. Vitamin C
content in the berries were significantly higher than the common vitamin C
sources like blackcurrant (1550 mg/kg ), lemon (650 mg/kg ), orange (500
mg/kg), grape (400 mg/kg), mango (250 mg/kg) and apple (150 mg/kg) (235).
Ascorbic acid in aqueous phase of plasma have significant role in AO
mechanism of body. It has many cellular activities that may be directly or
indirectly related to its AO properties. It has strong reducing property in
solution of pH> 4. In vivo most of the ascorbic acid is maintained in a
reduced state by other endogenous reductants. It has been demonstrated to be
an effective scavenger of superoxide, hydroxyl, and peroxyl radicals as well
as hydrogen peroxide and singlet oxygen. Ascorbic acid can directly act as
AO by reacting with aqueous radicals or indirectly by restoring the AO
activities of other AOs, like α-tocopherol. In studies with human plasma
lipids, ascorbic acid was found to be far more effective in inhibiting lipid
peroxidation initiated by peroxyl radical initiator than other AO components
such as protein thiols, urate, bilirubin and α-tocopherol (236).
Results & Discussion
110
3.1.7. Organic acid Composition of berries
SB Berries are highly acidic in nature. Total titrable acids in the berries
were found to be 3.6% in terms of malic acid. SB berry juice had a pH of 3.1.
Therefore the berries were analyzed in detail for their organic acid content.
Isolation of quinic acid: Quinic acid, a tetrahydroxy cyclohexanoic acid
(Figure 3.6) was isolated from the water extract of pulp by silica column
chromatography and identified by spectral techniques. Quinic acid thus
obtained was used as a reference compound for the HPLC analysis. Organic
acids along with high vitamin C content imparted considerable acidity to SB
berries. Detailed profiling of organic acids was attempted in this study.
HPLC profiling of organic acids: RP-HPLC-DAD analytical conditions for
the profiling of organic acids in SB berries were optimized for the first time.
Since the organic acids are polar in nature a RP C18 column was selected as
stationary phase. Phosphoric acid solution at pH 2.4 was used as mobile phase.
Low pH of the mobile phase is reported to lower the ionization of hydroxyl
and carboxyl protons and hence the mobility in non-polar stationary phase.
Highly acidic mobile phase inversely affect the life of column materials.
Therefore the acidity of mobile phase was selected so as to balance these two
opposite factors. HPLC equipped with DAD was used for the analysis. DAD
enables the recording of uv-visible spectra of analytes during the
chromatographic run. Spectro-chromatograms thus obtained is highly useful
for the identification of compounds and for assessing the purity of peaks.
Chromatographic conditions were optimized using the reference compounds
and the TR and uv spectra were recorded. Individual organic acids in the
samples were identified by comparing their TR and uv spectra with those of
reference compounds (Figure 3.7).
Results & Discussion
111
The composition of low molecular acids in the SB berries is given in
Table 3.5. The berries had 2.80% quinic acid in soft parts, along with malic
acid (1.60%), citric acid (0.16%) (Figure 3.6) and traces of fumaric and maleic
acids. The HPLC analysis showed quinic acid as the major organic acid in SB
berries. This finding is different from earlier reports of malic acid as the major
organic acid in SB berries (127). However, Beveridge et al; reported the
presence of quinic acid in processed SB juice. Higher amount of quinic acid a
biologically important organic acid signified the nutraceutical value of SB
berries (147). Sheng et al; identified quinic acid and its analogues as the active
molecules in the Cat’s claw extract, an effective medicinal preparation against
the chronic inflammations, and G-I dysfunctions like ulcer, tumors and
infections through in-vitro and in-vivo studies (236). SB berries are
recommended against G-I disorders in traditional medicinal systems. G-I
protective properties of SB berries might be attributed to the high content of
quinic acid. Detailed studies in this regard are required. Presence of reactive
hydroxyl and carboxylic acid groups makes quinic acid a promising starting
material for stereo specific cyclic organic compounds in synthetic chemistry.
Table 3.5. Vitamin C and organic acids content in H. rhamnoides fresh
berries (80% moisture) mean % ± SD.
Composition Amount
Vitamin C (mg/kg) 0.22 ± 0.02
Organic acids (%)
Quinic acid 2.80 ± 0.12
Malic acid 1.60 ± 0.09
Citric acid 0.16 ± 0.03
Total organic acids 4.56 ± 0.12
Results & Discussion
112
Figure. 3.6 Structures of major organic acids in SB berries
Figure 3.7 HPLC Chromatogram of organic acids in SB berry pulp.
O
HO
OH
O
OH
Malic acid
O O
OH
OH
OHO OH
Citric acid
OH
OH
HO
CO2H
OH
Quinic acid
QA: quinic acid, MA; malic acid, CA: Citric acid
Results & Discussion
113
3.1.8 Polyphenols
Phenolic compounds or polyphenols constitute one of the most
numerous and ubiquitously distributed group of plant secondary metabolites
with more than 8000 phenolic structures currently known. Natural polyphenols
can range from simple molecules (phenolic acids, phenyl propanoids,
flavonoids etc) to highly polymerised compounds (lignans, melanins, tannins
etc). These compounds play an important role in growth and reproduction,
provide protection against pathogens and predators and contribute towards
colour and sensory characters of plants. Polyphenols exhibit a wide range of
biological effects including anti-allergic, antimicrobial, anti-inflammatory,
anti-atherogenic, anticancer, hepatoprotective, cardioprotective and
vasodilatory actions (61,69,70,84,80). The varying chemical and biological
properties requisite the detailed chemical finger printing of this class for the
better understanding of phytochemistry of medicinal plants.
Previous reports suggest SB berries and leaves as a good source of
polyphenols and larger portion of their biological activities are attributed to
their polyphenol content. Reports on the detailed profiling of SB polyphenols
are rather limited. SB berries of Indian origin are particularly least studied on
this context. Therefore detailed profiling of polyphenols in the berry parts are
attempted in the present study.
Amount of polyphenols, in anatomical parts of SB berries were
quantified by using F-C reagent. F–C reagent method is widely used for the
quantification of phenolics in plant sources. In this method, a mixture of
molybdate and tungstanate in highly basic medium is used as the test mixture.
Reduction of molybdotungsatanate reagent to a blue product by phenolics is
the key step for this reaction.
Mo(VI) (yellow) Mo(V) (blue)
Results & Discussion
114
Calibration plot was obtained with gallic acid and results are expressed as
GAE (Table 3.6).
Pulp had 2.73% of phenolics in dry weight. Seed coat and kernel
respectively contained 3.95% and 8.58% phenolics. Amount of total phenolics
in the berries and seeds were deduced from these results. The whole berries
contained 3.62% phenolics whereas the seeds had 7.15% phenolics. The
values obtained for the phenolics in these berries are in accordance with the
previous reports on Chinese and Polish berries (158, 170).
Table 3.6. Total polyphenols, flavonols and PAs in H. rhamnoides berries
(dry mass). (Mean ± SD (n = 5).
Polyphenols
(%)
Flavonols
(%)
Proanthocyanidins
(%)
Pulp 2.73 ± 0.12 0.21 ± 0.05 1.26 ± 0.14
Seeds Coat 3.95 ± 0.04 0.02 ± 0.00 0.87 ± 0.10
Kernel 8.58 ± 0.32 0.53 ± 0.01 4.40 ± 0.22
Berries 3.62 0.24 1.67
Seeds 7.15 0.35 3.30
3.1.8.1 Flavonols
Flavonols are important class of polyphenols with significant and well-
documented biological properties. The amount of total flavonols, a major class
of flavonoids in the SB berries and leaves were quantified using aluminum
chloride reagent and summarized in Table 3.6. Aluminum chloride forms
chelating complexes with flavonols in quantitative manner resulting in a
bathochromic shift in band II absorption of flavonols (207). SB berry pulp,
Results & Discussion
115
seed coat and kernel were found to contain 0.21. 0.02 and 0.53% of flavonols.
Berries and seeds respectively had 0.24 and 0.35% flavonols.
According to the results of aluminum chloride quantification of
flavonols, flavonols contributed approximately 10% of total phenolics in SB
berries. Therefore detailed profiling of SB flavonols was attempted in this
study. Flavonoids are mainly occurring as their glycosides and esters in plants.
The variations in number, position and nature of glycosidic bonds and nature
of glycoside part result in great diversity in flavonoid glycosides. In order to
minimize the complexity, the SB flavonoid glycosides were hydrolyzed and
analyzed.
Repeated chromatographic separation of hydrosylate of kernel methanol
extract yielded two yellow colored solids with characteristics of flavonoids,
one of which was identified as quercetin by comparison with reference sample
and spectral data. The other was identified as isorhamnetin (Figure 3.8) by
spectroscopic studies. The compound showed uv-visible spectra of flavonols
with characteristic bathochromic shifts with NaOMe, AlCl3 and H3BO3. IR
showed characteristic absorption for hydroxyl groups. Mass spectra gave
molecular weight of 316 with characteristic flavonol fragmentation pattern.
1H-NMR spectra showed 5 aromatic protons at δ 8.31 (H-2’), 8.21 (H-6’), 7.37
( H-5’), 6.87 ( H-8), and 6.76 (H-6) and one methoxy group at δ 3.84. The
mass spectra (m/z 301, 151, 123, 108, 85, 57, 40) further confirmed the
structure of isorhamnetin.
RP-HPLC-DAD analysis of flavonols; RP-HPLC-DAD conditions for the
quantitative profiling of flavonoids were optimized using reference samples of
quercetin, isorhamnetin and kaempherol. Since the flavonoids are medium
polar in nature, RP C-18 column with an isocratic solution of methanol-water
mixture was used for the resolution of flavonoids. The individual SB
Results & Discussion
116
flavonoids were identified from their retention and spectral data and quantified
(Figure 3.9).
Quercetin, kaempherol and isorhamnetin (Figure 3.8) were identified as
the major flavonoids in the SB berries. Kernel (553 mg/100g) had the highest
amount of flavonoid followed by pulp (288 mg/100g) and seed coat (18
mg/100g) (Table 3.7). Both pulp and seeds had isorhamnetin as the major
flavonol followed by quercetin and kaempherol. 72-80% of the total flavonols
in the pulp and seeds was contributed by isorhamnetin, whereas quercetin
contributed 19-23%. The amounts of flavonols obtained for the present study
were in accordance with the previous reports (164,166, 167).
Table 3.7. Flavonoids in H. rhamnoides berries (mg/100g of dry weight).
Pulp Seed Coat Kernel
Quercetin 54.5 ± 8 4.2 ± 3 97.7 ± 11
Kaempherol 19.0 ± 2 0.8 ± 1 14.0 ± 8
Isorhamnetin 214.1 ± 8 13.4 ± 4 441.2 ± 7
Total 287.6 ± 17 18.4 ± 5 552.9 ± 13
Figure 3.8: Structures of major flavonols in SB berries
Flavonol R
Quercetin OH
Kaempherol H
Isorhamnetin OMe
O
O
R
OH
OH
OH
HO
Results & Discussion
117
Figure 3.9 HPLC-DAD chromatogram of flavonols in H. rhamnoides
berry pulp at 270 nm
1: quercetin, 2: Kaempherol, 3: Isorhamnetin
3.1.8.2 Proanthocyanidins (PA).
Tannins, formed by the condensation of simple phenolics, are another
important class of phenolics. Tannins are generally classified into hydrolysable
tannins and condensed tannins or proanthocyanidins (PA). Oligomeric and
polymeric flavon-3-ols are better known as PAs. The existence of PAs in
common foods including fruits, vegetables, cereals and wines, affect their
texture color and taste and may result beneficial and adverse nutritional
effects. Plant PAs posses a variety of physiological activities such as AO,
cardioprotection, anticancer and antihypertensive (168, 237-230). The greater
parts of these activities are governed by the chemical structure and degree of
polymerization of PAs.
2
Results & Discussion
118
Extraction of PA. The extraction efficiency of PA from plant materials
largely depends on their chemical nature, and solvent systems and extraction
conditions employed (240) which demand the optimization of extraction
conditions for the analysis of plant PA. PA comprises a wide range of
oligomeric and polymeric compounds with different sensitivity towards the
reagents used for their estimation (240). The diversity in PA and the presence
of other low molecular weight polyphenols like phenolic acids and different
classes of flavonoids make the selection of appropriate methods difficult for
the estimation of PA. In this study vanillin-HCl assay, a commonly used
method for the quantification of PA in plant extracts due to its specificity
towards flavonols and dihydrochalcones (240) was chosen. Since the reaction
of vanillin towards the polymeric PA is more specific and sensitive in
acidified methanol, the vanillin reagent was prepared in 5% HCl-methanol
and reactions were also carried out in the same solvent system (241).
The extractability of PA of SB berry pulp and seeds with methanol-
water and acetone-water mixtures and the effect of acidification were studied
and the results are presented in Figure 3.10. Introduction of water to methanol
and acetone resulted in considerable increase in the extractability of
polyphenols, particularly PA. It is obvious that, the permeability of plant
tissues is increased by the presence of water in extracting solvents, which
enables the better mass transport by diffusion. Acetone water mixtures
showed higher extractability, than corresponding methanol water mixtures (P
> 0.05). Acidification of solvent mixtures enhanced the extraction efficiencies
substantially in all samples studied here. 70% acetone water mixture
containing 1% acid was found as most effective solvent for the extraction of
PA from berry pulp, seed coat and kernel. The increased extractability of
polyphenols with the acidified solvent mixtures has been previously
demonstrated for beach pea (242). Higher extractability of polyphenols with
Results & Discussion
119
acidified solvent mixtures can be attributed to the acid catalyzed dissociation
of their bonds with polar fibrous matrices (243).
Crude PA extracts of SB berries for further studies were prepared using
the solvent mixtures as optimized. Crude PA extract of SB berry pulp,
prepared with 70% acetone water mixture containing 1% acid was analyzed
for its total phenolics and PA and the results are summarized in Table 3.8.
The extract had 6.75% phenolics and 3.24% PA. Based on these analyses, the
amount of PA in SB pulp was 1.17% (in dry matter). These values indicated
that nearly 50% of soluble phenolics in the berry pulp were contributed by
PA. Comparative evaluation of the results on PA from this study was not
attempted due to the lack of similar reports for SB berries of different origin.
Total PA content in the SB berry pulp (293 mg/100g) reported here was
comparable with that of cranberry (418 mg/100g), blue berry (256 mg/100g)
and blackcurrent (148 mg/100g), peach (67 mg/100g), green grapes (81
mg/100g) and black berry (27 mg/100g). SB berries could therefore be a
richer source for bioactive PA as compared to other edible fruits.
The seeds of SB berries were separated into seed coat and kernel, and
extracted with 70% acetone containing 1% acid. Crude PA extract of the
kernel contained significantly (P > 0.05) higher amount of polyphenols
(22.6%) and PA (11.6%) than berry pulp (Table 3.8). Total soluble phenolics
and PA in kernel were respectively 8.58 and 4.40%. Nearly half of the soluble
phenolics in kernel were also contributed by PA. The seed coat PA extract
had 30.9% polyphenols with 8.71% PA. The amount of soluble PA in seed
coat was respectively 3.95 and 0.87% respectively. Although the seed coat
extract contained higher amount of polyphenols than that in pulp and kernel
extracts (P > 0.05), the PA content was lower than that in those extracts.
Results & Discussion
120
Figure 3.10. Effect of different solvents on the extraction of PAs from SB
berry pulp, kernel, and seed coat.
0 20 40 60 80 1000
500
1000
1500
2000
2500
PA (mg/100g of dry matter
% of A in B
M in W
M in W + 1% HCl
A in W
A in W + 1% HCl
Pulp
M : methanol, W: water, A: acetone
Results & Discussion
121
0 20 40 60 80 100500
1000
1500
2000
2500
3000
3500
4000
4500PAs (mg/100g of dry matter
% of A in B
M in W
M in W + 1% HCl
A in W
A in W + 1% HCl
0 20 40 60 80 100
050100150200250300350400450500550600650700750800850900950
PAs (mg/100g of dry matter)
% of A in B
M in W
M in W + 1% HCl
A in W
A in W + 1% HCl
Kernel
Seed Coat
Figure 3.10. Continuing
M : methanol, W: water, A: acetone
Results & Discussion
122
Purification of PA extracts: Since the PAs constitute a major fraction of
polyphenols in SB berries, PAs were further purified and analysed. The crude
PA extracts were purified using sephadex (LH-20) gel column
chromatography. The yield of these purified fractions varied from 0.42 –
3.79% (Table 3.8). The total phenolic and PA in these purified extracts varied
from 56-69% and 59 - 69% respectively. 30 - 60% of total PA present in
crude extracts was found as recovered in purified extracts. These low
recovery values could be attributed to the possible elution of low molecular
weight PA with 95% ethanol (244). These purified extracts were used for the
evaluation of degree of polymerization.
Average degree of polymerization: PA are mixtures of oligomers and
polymers composed of flavan-3-ols mainly formed through C4 C8
and/or C4 C6 (B type) or linked through an ether bond between
C2 C7 (A type) and size of PA molecules is governed by their degree of
polymerization. Butler et al; suggests that the reaction of vanillin with PA in
acetic acid occurred only at the end groups without the depolymerisation of
PA and the ADP can be calculated as the ratio of absorbance of the
anthocyanins formed by the oxidative depolymerisation of PA to the
absorbance of vanillin - PA adduct (212).
The values obtained for ADP of SB PA as estimated from monomer to
polymer absorbance ratio showed seed coat PA showed the highest ADP of
8.2 followed by, berry pulp (7.4), and kernel (5.6). The ADP of kernel
obtained was lower than the value (DP = 12) reported by Fan et al 2007, by
the method of thiolysis, for the berries of Chinese origin (168). The
difference could be due to the difference in methodologies adopted or due to
the varietal or maturity differences of plant materials used. Lack of reports on
the amount and nature of PA in SB berries make the comparison of the results
obtained here difficult.
Results & Discussion
123
Table 3.8. Yield, total polyphenols and PAs in crude and purified PA
extracts and ADP of PAs in SB berries (mean ± SD, n=5).
Yield
(%)a
Total
polyphenolics
(%)b
Proantho
cyanidins
(PA) (%) c
ADP of PAs
Pulp
Crude PA
Extract 36.2 6.75 ± 0.11 3.24 ± 0.54 -
Purified PA
Extract 1.04 65.2 ± 0.54 68.04 ± 0.43 7.4 ± 0.6
Kernel -
Crude PA
Extract 38.3 22.6 ± 0.22 11.6 ± 0.91 -
Purified PA
Extract 3.79 57.5 ± 1.31 69.8 ± 3.21 5.6 ± 0.8
Seed Coat
Crude PA
Extract 9.7 30.92 ± 2.31 8.71 ± 0.98 -
Purified PA
Extract 0.61 56.05 ± 0.56 61.6 ± 2.38 8.2 ± 0.7
3.1.8.3 Phenolic Acids
Phenolic acids represent a major class of non-flavonoid plant
phenolics. Phenolic acids include two main groups namely, hydroxybenzoic
acid and hydroxycinnamic acid derivatives with different number and position
of hydroxylation and methoxylation in aromatic ring (Figure 3.11). Phenolic
acids are distributed as their free and bound forms in nature, more often
bound forms occur as their esters and glycosides. High molecular weight
phenolic acid glycosides constitute the class of hydrolysable tannins. There is
no comprehensive report on the phenolic acid composition in SB berries.
Therefore RP-HPLC-DAD analytical conditions for the profiling of SB
phenolic acids were optimized and anatomical parts of the berries were
analyzed in this study.
Arimboor et al JPBA Vol. 47 (1), 31-38 (2008)
Results & Discussion
124
Figure 3.11. Phenolic acids
Hydroxybenzoic acids (A) Hydroxycinnamic acids (B)
R1 R2 R3 R4 R1 R2
OH H H H salicylic acid H H cinnamic acid
H H OH H p-hydroxybenzoic acid H OH p-coumaric acid
H OH OH H 3,4-dihydroxybenzoic
acid
OH OH caffeic acid
H OH OMe H vanillic acid OH OMe ferulic acid
Standardization and validation of HPLC-DAD method
Chromatographic conditions: RP-HPLC-DAD conditions for the qualitative
and quantitative profiling of nine major phenolic acids viz; gallic acid,
protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, salicylic acid,
caffeic acid, p-coumaric acid, ferulic acid and cinnamic acid in SB berries
were optimized for the first time. Phenolic acids are polar in nature; therefore
different proportions of methanol and water were tried as mobile phase with
reverse stationary phase in this study. Variations in pH of mobile phase are
reported to have significant effect on the resolution and tailing of polar
compounds in reverse stationary phase. Presence of acids in mobile phase
can achieve better separation for phenolic acids, since it reduces the ionization
of phenol, phenolic hydroxyl and carboxylic acid groups (246, 247). Higher
concentration of acetic acid in mobile phase may result better separation,
which in turn would shorten the life of reverse phase column. A gradient of
Arimboor et al JPBA Vol. 47 (1), 31-38 (2008)
Co2H
R1
R2
CO2H
R1R2
R3
R4
A B
Results & Discussion
125
2% acetic acid in water and methanol was optimized as mobile phase and TR
of reference phenolic acids were obtained and the results are presented in
Figure 3.12 and Table 3.9.
DAD is capable of monitoring several wavelengths simultaneously, so
as to ensure that all uv-visible absorbing components are detected. HPLC-
DAD can therefore be used for recording uv-visible spectro-chromatograms
of compounds. Chromatograms at different wave lengths as obtained by
HPLC-DAD under the optimized conditions are given in Fig 3.12. The
wavelengths at which the compounds had maximum absorbance or maximum
peak/noise ratio were selected as their detection wavelength and are
summarized in Table 3.9. Vanillic acid and caffeic acid were found to be
eluted closely, but the differences in their absorption maxima could be
utilized for identifying them from each other. Vanillic acid had highest
absorbance at 270 nm and that was considerably lower at 329 nm, the
detection wavelength of caffeic acid. Therefore, these compounds could be
differentiated by comparing their chromatograms recorded at 270 nm and 329
nm (Fig 3.12).
System suitability tests: Regression equations were obtained by the external
standard method. A series of standard solutions of each of phenolic acids
were prepared and linearity between peak area and concentration was studied.
The linear range, regression equation and correlation coefficient of each
analytes were summarized in Table 3.9. All the components showed good
linearity (R ≥ 0.9893) in a relatively wide concentration range. The LOD and
LOQ of the method were evaluated and the values suggested that sensitivity
was satisfactory (Table 3.9).
` A solution mixture containing 50 µg/mL of each phenolic acids was
analyzed 5 times within a day to determine the intra day precision of the TR
a Arimboor et al JPBA Vol. 47 (1), 31-38 (2008)
Results & Discussion
126
and peak area by the method. The inter day precisions were determined by 6
analysis over two days for each phenolic acids (50 µg/mL). The relative
standard deviation (RSD) was taken as a measure of precision and the intra
day and inter day precisions for the TR and peak areas by the method are
summarized in Table 3.10. The RSD for the TR (< 1.8) and peak area (< 2.2)
were found to be low and inter day and intra day precisions were satisfactory
for all analytes. A reliable accuracy (97.4 – 103.9%) was also shown by this
method (Table 3.10). Reference phenolic acids were added to the phenolic
acid fractions and analyzed so as to determine the recovery and it was found
that the recovery of all phenolic acids was between 94.3 – 101.9% (Table
3.10). For stability test, the same sample solutions kept at 40 C was analyzed
every 12 h and the analytes were found to be rather stable with in 48 h.
Phenolic acid composition of H. rhamnoides berries: SB berries as pulp,
kernel and seed coat were analyzed for their free and bound phenolic acids
content. Phenolic acids in the berries and leaves were separated into three
fractions, viz; phenolic acids present in free form, phenolic acid liberated
from esters and phenolic acid liberated from glycosides and profiled by HPLC
for their phenolic acid composition. Peaks in the chromatograms were
identified by comparing with the TR and uv-visible spectra of reference
compounds and by spiking experiments. The amount of each analyte was
calculated from the corresponding calibration curve and represented as their
mean ± SD of three analyses (Table 3.11, Figure 3.13).
Composition of free and bound phenolic acids in the dry matter of
berry pulp is summarized in Table 3.11. Berry pulp contained 1068 mg/kg of
total phenolic acids, of which 58.8% was derived from phenolic glycosides.
Free and phenolic acids bound to esters respectively constituted 20.0 and 21.2
% of total phenolic acids. Gallic acid was identified as the predominant
phenolic acid both in free and bound forms, which accounted for 66.0% of
Arimboor et al JPBA Vol. 47 (1), 31-38 (2008)
Results & Discussion
127
total phenolic acids in pulp. Considerable amounts of protocatechuic acid
(136 mg/kg), ferulic acid (69 mg/kg), salicylic acid (54 mg/kg), p-
hydroxybenzoic acid (40 mg/kg) and p-coumaric acid (37 mg/kg) were found
to be present in pulp. Presence of vanillic acid (7 mg/kg), cinnamic acid (12
mg/kg) and caffeic acid (8 mg/kg) were also detected in pulp.
Seeds were separated into kernel and seed coat and analyzed for their
free and bound phenolic acids composition and the values on dry matter are
summarized in Table 3.11. The total phenolic acid content in kernel (5741
mg/kg) was higher than those in berry pulp and seed coat (Table 3.11).
Phenolic acids liberated from soluble esters (57.3% of total phenolic acids)
were the most abundant phenolic acid fraction in kernel. Free and bound
phenolic acids liberated from glycosidic bonds respectively contributed 8.4%
and 34.3% of total phenolic acids. The total phenolic acid content in the
kernel was two times higher than those reported for sesame and cotton seed,
and slightly lower than the values reported for rapeseed and canola meals, and
flax seed. Gallic acid (3441 mg/kg), protocatechuic acid (1330 mg/kg), p-
hydroxybenzoic acid (265 mg/kg), vanillic acid (368 mg/kg) and ferulic acid
(156 mg/kg) were the major phenolic acids present in kernel. Both free and
bound phenolic acid profile contained gallic acid as the predominant phenolic
acid, which contributed 59.9% of total phenolic acids in kernel. In addition to
this presence of cinnamic acid (82 mg/kg), p-coumaric acid (74 mg/kg) and
caffeic acid (25 mg/kg) were also detected in kernel.
The total soluble phenolic acids content in seed coat (448 mg/kg) was
lower than that in kernel and pulp (Table 3.11). Proportion of free phenolic
acids in total phenolic acids in seed coat was higher than that in kernel and
pulp. Phenolic acids bound to esters and glycosides respectively contributed
49.1% and 20.3% of total phenolic acids in seed coat. Gallic acid (230
mg/kg), protocatechuic acid (82 mg/kg), cinnamic acid (44 mg/kg), vanillic
Arimboor et al JPBA Vol. 47 (1), 31-38 (2008)
Results & Discussion
128
acid (39 mg/kg), p-hydroxybenzoic acid (26 mg/kg), ferulic acid (17 mg/kg)
and caffeic acid (10 mg/kg) were identified in seed coat. Protocatechuic acid
(43 mg/kg) was present as the major phenolic acid of the free phenolic acid
fraction, whereas gallic acid (200 mg/kg) was the major in bound phenolic
acid fractions in seed coat. Gallic and protocatechuic acids respectively
accounted for 51.3% and 18.3% of total phenolic acids in the seed coat.
Detailed reports on the composition of phenolic acids in anatomical
parts of SB berries are limited. Rosch et al 2003; reported the amount of gallic
acid (1.-2.6 mg/mL) and protocatechuic acid (2.1-2.9 mg/mL) in SB berry
juice (H. rhamnoides, collected from Germany) and their contribution to its
total AO capacity (164). Hakinen et al; reported the proportion of p-coumaric
acid, ferulic acid, p-hydroxybenzoic acid and ellagic acid in total phenolic
content of SB (H. rhamnoides) berries from Finland (248). In another report
on SB berries (H. rhamnoides) of Polish origin, salicylic acid was identified
as the predominant phenolic acid both in free and bound forms by GC-MS
analysis (170). Presence of gallic acid in SB leaves is previously reported
(249). In these reports, the SB berries were analyzed as whole for the phenolic
acid composition and their distribution of phenolic acids among the
anatomical parts of berries as done in the present study was not discussed in
detail. In our study free and bound phenolic acid composition of pulp, seed
coat and kernel of SB berries were separately studied for the first time. The
major fraction (Approx. 70%) of phenolic acids in SB berries was found to be
concentrated in the seeds. The report of salicylic acid as the predominant
phenolic acid in SB berries from Poland (170) is contrary to the present study,
where gallic acid was found as the major phenolic acid both in pulp and seeds
of berries of Indian origin. The berry pulp in this study contained only 54
mg/kg of salicylic acid, which contributed 5.06% of total phenolic acids in
pulp. These results warrant the detailed studies on the phenolic composition
of SB berries from different varieties and geo-climatic regions.
Arimboor et al JPBA Vol. 47 (1), 31-38 (2008)
Results & Discussion
129
Table 3.9. Detection wave length, TR and linear regression data of phenolic acids.
a x : peak area, y : concentration (mg/mL).
b Detection was expressed as LOD = 3.3s/a, where a is the slope, and s is residual standard deviation of regression line
Compound
Detection
wavelength
(nm)
TR
(Min)
Regression equationa
Linear range
(µg mL-1)
Correlation
coefficient
LODb
(µg mL-1)
Gallic acid
280
8.4
Y = 1.298 X 105 x + 187
10.8– 150.2
0.9992
1.20
Protocatechuic acid
260
16.8
Y = 0.991 X 105 x - 1101
8.0–220.4
0.9995
0.72
p-hydroxybenzoic
acid
255
25.6
Y =1.894 X 105 x - 548
12.5 - 175
0.9924
2.78
Vanillic acid
260
31.8
Y = 1.374 X 105 x + 413
13.2 - 150
0.9893
1.97
Salicylic acid
300
42.5
Y = 0.844 X 105 x + 1332
12.4–175.2
0.9961
2.87
Cinnamic acid
275
49.8
Y = 1.282 X 105 x + 990
10.1–166.8
0.9971
1.77
Caffeic acid
325
32.4
Y = 2.307 X 105 x + 14489
6.6–208.5
0.9939
1.04
Ferulic acid
310
39.2
Y = 2.357 X 105 x – 15307
5.0–220.0
0.9981
1.65
p-Coumaric acid
325
40.8
Y = 2.443 X 105 x + 1495
10.4–240.8
0.9903
0.65
Arimboor et al JPBA Vol. 47 (1), 31-38 (2008)
Results & Discussion
130
Table 3.10. Precision, accuracy and recovery of phenolic acids.
Compounds
Precision (RSD %)a
Accuracy
(%)d
Recoverye (%)
Intra dayb
Inter dayc
RT
peak area
RT
peak area
Gallic acid
0.92
1.62
1.12
2.05
99.6 ± 0.8
92.5 ± 1.2
Protocatechuic acid
0.72
1.71
1.33
1.98
102.3 ± 1.0
94.3 ± 2.1
p-hydroxybenzoic acid
0.85
1.25
1.28
1.79
98.1 ± 0.9
102.5 ± 1.9
Vanillic acid
1.01
1.38
1.78
2.23
101.7 ± 2.1
98.2 ± 1.8
Salicylic acid
1.09
1.72
1.12
1.87
103.9 ± 0.4
95.9 ± 1.1
Cinnamic acid
1.13
1.41
1.36
1.08
98.9 ± 0.8
98.7 ± 1.6
Caffeic acid
0.97
0.82
1.61
0.85
98.8 ± 1.9
98.1 ± 1.9
Ferulic acid
0.78
1.66
1.19
0.97
100.6 ± 1.4
96.6 ± 2.2
p-Coumaric acid
1.09
1.34
1.06
1.28
97.4 ± 0.6
97.2 ± 1.8
a RSD% = ((SD/mean) X 100), b n = 5, c n = 6. d Mean (Found/nominal)X100% (n=6) ± SD, at medium analyte concentration of 50 µg
mL-1. e Mean ± SD (n = 3).
a Arimboor et al JPBA V
ol. 47 (1), 31-38 (2008)
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Results & Discussion
131
Table 3.11. Free and bound phenolic acids in berry pulp, kernel and seed coat (mg/kg of dry matter) (mean ± SD (n = 3).
Phenolic acids
Hydroxybenzoic acid derivatives
Hydroxycinnamic Acid Derivatives
Total
GA
Pro-CA
p-HBA
V A
S A
CA
P-CA
FA
Caf. A
Pulp
Free
88 ± 5.7
33 ± 2.5
8 ± 1.8
5 ± 0.4
46 ± 1.2
7 ± 0.2
11 ± 1.8
13 ± 2.9
3 ± 0.4
214 ± 9.6
Bound
Esters
152 ± 1.0
23 ± 0.6
8 ± 3.2
1 ± 0.4
5 ± 0.8
5 ± 0.2
12 ± 0.3
18 ± 0.1
2 ± 0.3
226 ± 4.3
glycosid
es
465 ± 0.3
80 ± 0.1
24 ± 1.0
1 ± 0.1
3 ± 0.3
-
14 ± 4.8
38 ± 1.9
3 ± 0.1
628 ± 2.5
Total
705
136
40
7
54
12
37
69
8
1068
Kernel
Free
329 ± 16.2
99 ± 12.1
28 ± 3.9
5 ± 0.2
ND
- 5 ± 0.3
5 ± 0.4
10 ± 0.8
3 ± 0.3
484 ± 21.6
Bound
Esters
2198 ± 221.4
690 ± 77.7
144 ± 5.7
47 ± 7.5
ND
52 ± 3.2
65 ± 12.7
81 ± 4.6
12 ± 0.9
3289 ± 236.9
glycosid
es
914 ± 18.0
541 ± 12.1
93 ± 6.7
316 ±
34.3
ND
25 ± 1.0
4 ± 0.0
65 ± 2.2
10 ± 9.4
1968 ± 123.3
Total
3441
1330
265
368
ND
82
74
156
25
5741
Seed
coat
Free
30 ± 5.3
43 ± 10.8
17 ± 4.7
12 ± 4.5
ND
19 ± 1.2
ND
15 ± 2.2
1 ± 0.4
137 ± 5.6
Bound
Esters
164 ± 8.3
27 ± 0.6
7 ± 0.8
5 ± 0.8
ND
11 ± 0.1
ND
2 ± 0.2
4 ± 0.1
220 ± 5.6.
glycosid
es
36 ± 2.4
12 ± 0.7
2 ± 0.3
22 ± 0.3
ND
14 ± 0.7
ND
- 5 ± 0.2
91 ± 2.5
Total
230
82
26
39
ND
44
ND
17
10
448
Leaves
Free
689 ± 13.6
16 ± 1.8
144 ± 6.6
17 ± 1.7
ND
122 ± 0.5
1 ± 0.0
44 ± 2.9
18 ± 1.9
1051 ± 30.2
Bound
Esters
2899 ± 23.2
19 ± 1.6
36 ± 4.3
6 ± 0.0
ND
54 ± 5.2
ND
119 ± 1.6
ND
3133 ± 17.7
glycosid
es
634 ± 0.2
15 ± 0.4
67 ± 0.7
14 ± 0.9
ND
62 ± 1.8
ND
12 ± 0.5
ND
804 ± 5.9
Total
4222
50
247
37
ND
238
1
175
18
4988
Arimboor et al JPBA Vol. 47 (1), 31-38 (2008)
Results & Discussion
132
Figure 3.12 HPLC-DAD chromatograms of phenolic acids at different
wavelengths.
Arimboor et al JPBA Vol. 47 (1), 31-38 (2008)
Results & Discussion
133
Figure 3.13. HPLC-DAD chromatograms of phenolic acids in H.
rhamnoides kernel extract at different wavelengths.
Arimboor et al JPBA Vol. 47 (1), 31-38 (2008)
Results & Discussion
134
This is the first comprehensive report on the chemical evaluation of H
rhamnoides berries of Indian origin. Detailed studies with large number of
samples of different species and from different geo-climatic region are
required for the complete chemical finger printing of Indian SB. Comparison
of berries belonging to major species of SB in India is attempted in section
3.2
Results & Discussion
135
3.2. Phytochemical Profiling of Berries of Major
SB Species in Indian Trans-Himalayas
In India, SB is abundantly grown in the Trans-Himalayan cold deserts
such as Ladakh, Lahaul, and Spiti at altitudes from 2500 to 4500 m. The
Ladakh region extends between 32° 15’–36° N latitude and 75° 15’–80° 15’ E
longitude; Spiti and Lahaul lie between 31° 44’–32° 59’ N latitude and 76°
46’–78° 41’ E longitude. H. rhamnoides, H. salicifolia, and H. tibetana are
the predominant SB species found in Indian Himalayas. H. rhamnoides is
widely distributed in the Trans-Himalayan region, but H. salicifolia and H.
tibetana have limited distribution and are observed only in the Lahaul and
Spiti regions, respectively (130,138,250). The SB-growing areas in Trans-
Himalayas host unique geoclimatic conditions of high altitude coupled with
extreme temperature variations (–40 to 30°C), low precipitation, and low
oxygen in air. In these cold deserts plants are under extreme climatic stress
and are expected to have a distinct phytochemical profile. No systematic
studies have been carried out from this perspective on Himalayan SB berries,
particularly on H. salicifolia and H. tibetana, which are exclusive to high
altitudes of Himalayas. This chapter deals with the chemical composition of
berries belonging to three major SB species (H. rhamnoides, H. salicifolia,
and H. tibetana) from different locations in Indian Trans-Himalayan region.
Nutritional quality of these berries was compared in terms of their proximate
composition, polyphenol, flavonoid, and vitamin C contents and lipid profile.
Ranjith et al JAOCS, 83, 359-364 (2006).
Results & Discussion
136
Table 3.12; Seed content and proximate composition of SB berries of H .rh
amnoides, H. salcifolia and H
.
tibetana species from India (Mean ± S D, n=3).
SB species
Geographic
location
Seed
(%)
Moisture
(%)
Pulp oil
(%)
Ash
(%)
Protein (%)
H.
rhamnoides
Lahaul-1
10.4
74.4±0.5
3.08±0.22
1.05±0.02
2.25±0.62
Lahaul-2
8.1
74.4±0.4
2.99±0.32
0.79±0.14
1.86±0.59
Lahaul-3
9.7
72.7±0.2
3.04±0.26
0.93±0.19
2.11±0.45
H.
rhamnoides
Spiti-1
7.9
76.9±0.7
3.38±0.45
0.60±0.29
2.17±0.49
Spiti-2
7.4
74.6±0.5
3.14±0.23
0.73±0.09
2.15±0.34
Spiti-3
9.3
67.2±1.3
3.38±0.17
0.99±0.14
2.44±0.19
H.
rhamnoides
Ladakh-1
9.8
72.3±0.9
3.60±0.15
0.89±0.12
2.12±0.25
Ladakh-2
8.5
72.1±0.5
3.52±0.31
0.79±0.11
1.99±0.23
Ladakh-3
8.6
73.4±0.4
3.61±0.25
0.92±0.09
2.34±0.39
H. salicifolia
Lahaul-1
3.4
85.8±1.0
1.49±0.20
0.26±0.06
1.13±0.29
Lahaul-2
3.7
82.3±0.9
1.53±0.15
0.40±0.08
1.54±0.38
Lahaul-3
4.1
83.5±0.6
1.65±0.28
0.39±0.02
1.62±0.32
H.
tibetana
Spiti-1
9.0
74.5±0.4
2.25±0.56
1.02±0.10
3.09±0.13
Spiti-2
8.8
73.2±1.1
2.45±0.32
0.96±0.09
2.98±0.22
Spiti-3
8.7
72.9±0.9
2.50±0.19
0.87±0.12
2.96±0.29
Ranjith et al JAOCS, 83, 359-364 (2006).
Results & Discussion
137
3.2.1 Proximate composition of berries.
The proximate composition of fresh berries of the three different species is
summarized in Table 3.12. The moisture content of fresh berries of H. tibetana
and H. rhamnoides was in the range of 67–77%, and in H. salicifolia was 82–
86%. Seeds constituted 7.4–10% of the fresh berries in H. tibetana and H.
rhamnoides and 3.4–4.1% in H. salicifolia. This showed H. salicifolia had higher
proportion of juicy pulp than the other two species. In these berry samples, the
oil content of the pulp of H. rhamnoides contained higher amounts of oil (3.0–
3.6% of fresh berries) than for H. tibetana (2.2–2.5%) and H. salicifolia (1.5–
1.6%). The ash content of H. rhamnoides and H. tibetana (0.60 – 1.05% of
berries) was also higher than in H. salicifolia (0.26 – 0.40%). Total protein
content in the pulp of the berries was found to be higher for H. tibetana (2.96–
3.09% of fresh berries) than for H. rhamnoides (1.86–2.44%) and H. salicifolia
(1.13–1.62% of berries). In general, proximate composition of SB berries varied
from species to species. Among the studied samples only H. rhamnoides was
obtained from different geographical locations. Not much variation was observed
in the proximate composition among H. rhamnoides berries collected from
different geographical locations.
3.2.2 Vitamin C content in berries
SB berries are well known for their extraordinarily high levels of vitamin
C, which is considered as an important quality parameter of SB berries. Several
authors have reported high vitamin C content in SB berries (3600 – 25000
mg/kg) (127). The studied berries of Indian origin were also found to contain
high amount of vitamin C with wide range (1444 – 29840 mg/kg) (Table 3.13).
Vitamin C content in the berries of H. salicifolia (22,979–29,840 mg/kg) and H.
tibetana (8,789–9,279 mg/kg) were much higher than that in the common species
Ranjith et al JAOCS, 83, 359-364 (2006).
Results & Discussion
138
H. rhamnoides (1,444–4,877 mg/kg) (Table 3.13). Vitamin C content in the H.
salicifolia was particularly interesting due to its significantly high value.
Extensive variations in vitamin C content in H. rhamnoides berries have
been reported among individuals, populations and subspecies. The vitamin C
concentration ranging from 28 to 310 mg/100g of berries has been reported in the
European ssp. rhamnoides (251). Wide ranges of vitamin C content have been
reported for berries of Russian cultivars belonging to ssp. mongolica (40 to 300
mg/100g of berries) and ssp. fluviatilis (460 to 1330 mg/100g of berries) (252).
Chinese cultivars of ssp. sinesnsis have a vitamin C content of 200 to 2500
mg/100g of berries (253-255).
3.2.3. Polyphenol content in berries:
Total phenolics in the berries was determined using F-C reagent method
and tabulated in Table 3.13. The amount of the phenolics in pulp of the berries
ranged from 3950 to 6719 mg/kg of fresh berries. Among the studied samples H.
salcifolia berry pulp contained highest amount of phenolics (5913 – 6719 mg/kg)
followed by H. tibetana (5719 – 5893 mg/kg) and H. rhamnoides (3956 – 5728
mg/kg). H. rhamnoides berries from Spiti region had lower amount of phenolics
in their pulp than those from Ladakh and Lahaul regions. The values obtained for
the phenolics content in Indian SB berries was in accordance with the H.
rhamnoides berries of Chinese and Polish origin (158, 170). There are no reports
on the phenolics content of H. salcifolia and H. tibetana berries.
Ranjith et al JAOCS, 83, 359-364 (2006).
Results & Discussion
139
Table 3.13. Vitamin C, polyphenol and flavonol content of SB berries of
H.rhamnoides, H. salcifolia and H. tibetana Species from India (Mean ± S D, n=3).
SB species Geographic
location
Vitamin C
(mg/kg)
Polyphenols
(mg/kg)
Flavonols
(mg/kg)
H. rhamnoides Lahaul-1 2924 ± 36 5646 ± 16 308 ± 12
Lahaul-2 2305 ± 29 5213 ± 32 285 ± 21
Lahaul-3 4877 ± 19 5138 ± 19 212 ± 49
H. rhamnoides Spiti-1 1866 ± 17 3956 ± 21 122 ± 20
Spiti-2 1444 ± 36 4132 ± 39 156 ± 17
Spiti-3 1666 ± 23 4018 ± 17 180 ± 9
H. rhamnoides
Ladakh-1 2145 ± 53 5319 ± 51 251 ± 25
Ladakh-2 2568 ± 42 5728 ± 19 291 ± 17
Ladakh-3 2425 ± 36 5219 ± 72 261 ± 22
H. salicifolia
Lahaul-1 27692 ± 91 6019 ± 35 428 ± 51
Lahaul-2 29840 ± 185 5913 ± 42 353 ± 16
Lahaul-3 22979 ± 78 6719 ± 33 398 ± 9
H. tibetana
Spiti-1 9279 ± 79 5893 ± 47 392 ± 12
Spiti-2 8789 ± 65 5719 ± 35 401 ± 15
Spiti-3 89845 ± 45 5811 ± 27 342 ± 14
Ranjith et al JAOCS, 83, 359-364 (2006).
Results & Discussion
140
3.2.1. Flavonoids: Aluminium chloride forms complexes with flavonols in a
quantitative manner resulting a bathchromic shift in band II absorption of
flavonols (207). This property is widely used for the high through put
quantification of flavonols. Flavonols contribute a major fraction of polyphenols
in berry pulp. Total flavonol content in the berry pulp was determined using
aluminium chloride method and tabulated in Table 3.13. H. salicifolia (353 –
428 mg/kg) and H. tibetana (342 – 401 mg/kg) had slightly higher amount of
flavonols than in H. rhamnoides (122 – 308 mg/kg). H. rhamnoides collected
from Spiti region had slightly lower amount of flavonols than those from Lahaul
and Ladakh. The amount of flavonols (122–428 mg/kg) in the berries of these
three species was also found to be slightly higher than the reported amounts of
flavonols in cranberry (100–263 mg/kg), bog whortleberry (184 mg/kg),
lingonberry (74–146 mg/kg), blackcurrant (115 mg/kg), and crowberry (102
mg/kg).
The results presented here indicate that the chemical profile of H.
salicifolia, with a lower seed content, lower levels of fatty matter, and high levels
of vitamin C and flavonoids, differed perceptibly from the other two. The
chemical properties of H. salicifolia and H. tibetana have not been well
documented until now.
3.2.4. Composition of pulp oil.
Nutritional quality of the SB pulp oil is attributed to its high carotenoid
and tocopherol content along with unique fatty acid profile. Nutritional quality of
the pulp oil from the three species of SB was evaluated in terms of their chemical
composition.
Ranjith et al JAOCS, 83, 359-364, (2006).
Results & Discussion
141
Table: 3.14. FA composition of pulp oils of SB berries from H
.rhamnoides, H. salcifolia and H
. tibetana Species from
India (Mean ± S D, n=3).
S.B species
Geographic
location
FA (wt%)
14:0
16:0
16:1
18:0
18:1
18:2
18:3
H.
rhamnoides
Lahaul-1
0.5 ± 0.1
28.4 ± 1.1
50.3 ± 1.2
0.6 ± 0.0
11.3 ± 1.7
8.1 ± 0.3
1.3 ± 0.9
Lahaul-2
0.6 ± 0.0
29.4 ± 0.7
50.1 ± 1.1
0.7 ± 0.1
9.7 ± 0.6
7.9 ± 0.2
1.6 ± 0.7
Lahaul-3
0.5 ± 0.0
28.3 ± 1.1
49.7 ± 0.9
0.5 ± 0.0
11.5 ± 1.6
8.5 ± 0.4
1.7 ± 0.8
H.
rhamnoides
Spiti-1
0.6 ± 0.2
35.8 ± 1.2
45.6 ± 2.3
0.5 ± 0.1
9.8 ± 1.5
8.2 ± 0.2
0.8 ± 0.1
Spiti-2
0.3 ± 0.1
35.8 ± 1.1
45.6 ± 1.9
Tr
9.8 ± 1.3
8.2 ± 0.8
0.8 ± 0.1
Spiti-3
0.6 ± 0.0
30.7 ± 2.6
51.8 ± 0.9
Tr
12.3 ± 0.6
5.9 ± 1.0
0.4 ± 0.0
H.
rhamnoides
Ladakh-1
0.4 ± 0.0
33.8 ± 1.5
46.4 ± 2.0
1.0 ± 0.1
13.4 ± 0.94
5.4 ± 1.3
0.4 ±1.3
Ladakh-2
0.9 ± 0.1
31.2 ± 1.4
49.1 ± 3.1
0.8 ± 0.0 13.9 ± 3.49
4.6 ± 0.6
Tr
Ladakh-3
0.8 ± 0.1
30.9 ± 1.3
45.6 ± 2.6
0.9 ± 0.0
16.8 ± 1.73
5.8 ± 0.4
0.8 ± 0.4
H.
salicifolia
Lahaul-1
0.3 ± 0.1
29.0 ± 3.1
32.9 ± 0.5
0.1 ± 0.0
17.6 ± 0.5
16.1 ± 0.9
0.6 ± 0.1
Lahaul-2
0.1 ± 0.0
28.9 ± 0.8
41.2 ± 1.2
0.7 ± 0.1
12.6 ± 1.1
14.3 ± 1.8
1.0 ± 0.0
Lahaul-3
0.2 ± 0.1
28.0 ± 1.9
35.4 ± 3.0
0.6 ± 0.2
13.1 ± 1.3
15.0 ± 1.3
0.8 ± 0.2
H.
tibetana
Spiti-1
1.1 ± 0.2
25.7 ± 0.5
32.1 ± 4.2
0.5 ±0.1
26.0 ± 1.9
9.3 ± 1.1
5.2 ± 1.0
Spiti-2
0.9 ± 0.3
28.5 ± 2.4
34.1 ± 2.3
0.4 ± 0.0
22.2 ± 2.8
9.2 ± 0.5
3.5 ± 0.6
Spiti-3
0.8 ± 0.1
26 ± 1.8
36.0 ± 1.4
0.4 ± 0.2
23.5 ± 2.4
8.3 ± 0.8
5.6 ± 0.9
Ranjith et al JAOCS, 83, 359-364 (2006).
Results & Discussion
142
3.2.4.1. FA composition. The FA compositions of pulp oil of the three species of
berries are summarized in Table 3.14. In the pulp oil, the dominating FAs were
palmitoleic, palmitic, oleic, linoleic, and linolenic acids. The highest variations
among the three species were observed in the proportion of palmitoleic acid (32–
50%). H. rhamnoides contained the highest proportion of this rare FA in the pulp
oil (45–50%). Variation in the next highest FA, palmitic acid, was in the range of
25–36% in the pulp oil from the three species. The proportion of oleic acid (22–
26%) in pulp oil of H. tibetana was higher than that of H. salicifolia (13-18%)
and H. rhamnoides (10–17%). The proportion of palmitoleic acid in the pulp oils
of the studied berries was found to have a negative correlation with that of oleic
acid. The average amount of palmitoleic acid was highest in H. rhamnoides
(48.2%) followed by H. salicifolia (36.5%) and H. tibetana (34.0%) pulp oils and
the amount of oleic acid followed the reverse order (11.7, 14.4 and 23.6%
respectively). The proportion of palmitoleic acid correlates negatively with that
of oleic acid. The highest level of palmitoleic acid (up to 54%), and the lowest
level of oleic acid (as low as 2%) are reported for subsp. mongolica (145,148).
In the present study the proportion of ALA was found to be slightly higher in H.
tibetana (3.5–5.6%) than that in the other two species. Traces (>1%) of myristic
and stearic acids were also present in all three species. Trace amounts of
arachidic acid (20:0) were observed in pulp oil of H. salicifolia (data not shown).
This is the first report on the FA profile of SB berries of Indian origin.
The variations in the FA profile of pulp oils from the berries belonging to
different species and geographical regions are previously reported by many
authors (148,150,173,256,257,). Large variations were observed in the
proportions of palmitoleic, palmitic and oleic acids. For example Kallio et al;
reported that H. rhamnoides berries of ssp. mongolica contained less oleic acid
Ranjith et al JAOCS, 83, 359-364 (2006).
Results & Discussion
143
(4.6 v/s 20.2%) and more palmitic (33.9 v/s 27.4%) and palmitoleic (32.8 v/s
21.9%) acids than those of ssp. sinesis (171). According to Abid et al; H.
rhamnoides berries from Northern Pakistan contained almost equal amounts of
palmitic (34.5%) and palmitoleic (33.4%) acids as predominant FA (175). The
results of the present study showed palmitoleic acid as the predominant FA in the
pulp oil of Indian berries with extreme variations from 32 to 50%. In considering
the therapeutic value of this rare FA, these results indicate the potential of
breeding and industrial application to evolve palmitoleic acid rich varieties.
3.2.4.2. Carotenoids. Carotenoids in the soft parts impart beautiful yellow
orange color to SB berries. The amounts of total carotenoids in the pulp oils of
berries were quantified and expressed in terms of β-carotene (Table 3.15). The
amount of carotenoids in pulp oils of these species varied from 700 to 3500
mg/kg. The carotenoid content of pulp oils of H. rhamnoides (2350 – 3420
mg/kg) and H. tibetana (2693 – 3166 mg/kg) were similar, and H. salicifolia had
the lowest amount (692 – 840 mg/kg). The greenish yellow color of H. salicifolia
berry itself indicated its low carotenoid content. Carotenoid content in the pulp
oil of H. rhamnoides berries from Ladakh (2350 – 2650 mg/kg) was slightly
lower than that from the Lahaul (2660 – 3420 mg/kg) and Spiti (2576 – 2820
mg/kg) regions. The carotenoid content in H. rhamnoides was comparable with
the values reported for the berries of Chinese origin (129). However, authentic
reports on the carotenoid content in H. tibetana and H. salicifolia from other
geographical areas are not available. β-Carotene as the major component in SB
pulp oils as well as α-carotene, γ-carotene, dihydroxy carotene, lycopene, and
xeaxanthin has been reported (258). Carotenoid content in the berries are
subjected to extreme variation; differences up to 10-fold has been reported even
with in the same natural population and subspecies. Levels of total carotenoids
from 1-120 mg/kg of fresh berries have been reported in the literature.
Ranjith et al J AOCS, 83, 359-364 (2006).
Results & Discussion
144
3.2.4.3. Tocopherols/tocotrienols.
Tocols in pulp oil were quantified by HPLC, and the results are given
Table 3.15. The amount of tocols in pulp oil varied from 600 to 1800 mg/kg
among the three species studied. H. salicifolia contained the least amount of
tocols (666 – 902 mg/kg), as compared with H. rhamnoides (1301 – 1788 mg/kg)
and H. tibetana (1368 – 1546 mg/kg). H. rhamnoides berries from the Lahaul
region showed a slightly higher content of tocols than those from Spiti and
Ladakh. The relative proportion of individual tocols was almost identical in the
three species studied here with α-tocopherol predominating (40–60%).
Tocopherols constituted about 70 to 80% of total tocols, whereas
tocotrienols accounted for 20 to 30%. The proportion of the other major isomers,
α-tocotrienol and γ-tocopherol, was 5–25 and 4–16%, respectively. γ-Tocotrienol
and δ-tocopherol together represented 10–25% of total tocols. Other isomers of
tocols were present in trace amounts. The total amount of tocopherols in these
berries, except for H. salicifolia, was in accordance with the values reported from
China and Poland (171,157). The tocopherol profile of Indian berries differed
from the values reported for the Poland varieties in the proportion of δ -
tocopherol. More resemblance in tocopherol profile of H. rhamnoides cultivars
between Indian and Chinese origin might be due to the geo-climatic closeness in
growing areas. Comparison of the tocopherol profile of berries of H. salicifolia
and H. tibetana species with those of other geographic areas is not attempted
here due to the lack of authentic reports. SB berries from the Indian region of the
Himalayas were therefore a very rich source of tocols and were comparable with
those of berries from other regions in the case of H. rhamnoides. It is well known
that, as AOs, tocols, particularly tocotrienols, modulate diseases such as
atherosclerosis, diabetes, cancer, ageing, and the like
Ranjith et al J AOCS, 83, 359-364 (2006).
Results & Discussion
145
Table-3.15: Carotenoids, tocopherols and tocotrienols composition of pulp oils of SB berries of H. rh
amnoides, H.
salcifolia and H
. tibetana species from India (mg/kg of pulp oil) (Mean ± S D, n=3) .
SB. Species
Geograph
ic
location
Carotenoids
Tocopherols/tocotrienols
α ααα T1
α ααα T3
β T1
γ γγγ T1
γ γγγ T3+ δ δδδT1
δ δδδT3
Total
H.
rhamnoides
Lahaul-1
2660 ±82
859±40
293±24
16±5
152±21
362±45
12±2
1694±61
Lahaul-2
3420 ±102
1068±49
227±16
12±2
165±13
288±21
17±2
1788±41
Lahaul-3
2940 ±42
773±28
148±28
19±4
149±28
344±21
16±4
1564±67
H.
rhamnoides
Spiti-1
2576 ±108
1006±26
183±22
18±3
170±35
228±24
10±3
1615±76
Spiti-2
2820 ±62
1046±71
164±34
9±4
120±22
117±23
16±3
1494±76
Spiti-3
2706±35
679±54
247±28
11±4
121±20
241±14
17±1
1318±59
H.
rhamnoides
Ladakh-1
2400 ±60
954±73
95±15
27±6
126±5
187±11
14±2
1394±87
Ladakh-2
2350 ±22
858±73
120±27
22±4
82±10
201±22
15±1
1301±80
Ladakh-3
2650 ±61
903±50
109±27
21±5
90±5
208±35
17±1
1348±93
H.
salicifolia
Lahaul-1
801 ±25
354±15
46±7
5±2
94±9
291±18
8±1
799±23
Lahaul-2
840 ±42
517±30
124±23
4±1
57±18
183±12
18±6
902±35
Lahaul-1
692 ±52
390±22
89±12
9±2
33±6
128±35
18±1
666±62
H.
tibetana
Spiti-1
3166 ±32
867±46
127±17
15±3
86±16
431±46
19±4
1546±47
Spiti-2
2693 ±41
895±67
95±7
18±3
62±18
363±30
17±5 1448±109
Spiti-3
2840 ±18
864±13
85±11
19±2
64±16
336±33
12±4
1368±25
Ranjith et al J AOCS, 83, 359-364 (2006).
Results & Discussion
146
Chemical composition of SB berries largely depends on their
origin/ssp., geo-climatic conditions and agronomic practices (131).
Systematic mapping of chemical composition of SB berries of different
varieties and origins are still lacking. The results of the present study showed
that the pulp oil of two Indian SB berries (H. rhamnoides and H. tibetana)
were rich sources of bioactive lipophilic compounds, i.e., carotenoids and
tocopherols, and had a distinct FA profile, with palmitoleic acid as the major
FA. The presence of palmitoleic acid as the predominant FA is unique among
oils of plant origin, and this observation verified previous reports based on
Chinese varieties (131,148,171).For the first time the distinct nature of the
berries of H. salicifolia, which is only found in the Himalayan region, was
revealed, including a high content of hydrophilic components such as
flavonols and vitamin C and a very low content of oil and lipophilic
components such as carotenoids and tocopherols. The high nutritional quality
of berries of H. tibetana, which is found only in India, is reported here for the
first time. A high content of tocopherols and carotenoids, with high amount of
palmitoleic acid and other bioactive molecules, enhances the value of SB pulp
oil as a health supplement. Reports on the lipophilic constituents of SB berries
from China, Canada, Poland, and Russia are available. However, no authentic
data on the SB berries grown in the Indian region of the Himalayas have been
reported. The cold desert conditions in which Indian SB is grown may have
an influence on the phytochemical profile, as shown in this preliminary study.
Detailed studies with large number of samples representing different varieties
and geoclimatic conditions are required for the complete finger printing of
Indian SB berries.
Ranjith et al JAOCS, 83, 359-364 ( 2006).
Results & Discussion
147
3. 3. Processing of Fresh Sea Buckthorn Berries
Indian Himalayas hosts world’s second or third largest cultivation of
SB. Even though so far, no attempt has been made to utilize SB berries grown
in this region. An attempt has been made here for the first time to develop a
process for the production of high quality pulp oil and oil free clear juice with
maximum retention of bioactive phytochemicals. The seeds obtained as a
byproduct were subjected to SC-CO2 extraction for superior quality oil. Three
batches of SB berries were processed at pilot scale and process parameters
were analysed. The present study also reports a detailed chemical evaluation
of process streams and products from Indian SB berries for the first time.
3.3.1. Process development
The schematic diagram of the process is given in Figure 2.1.
Dewatering is the most critical operation of this process, the efficiency of
which determines the product yield and quality. A continuous dewatering
screw press, designed in our laboratory for fresh spices with high moisture
content (700–800 g/kg), was used for this operation. The fresh berries used
here had an average seed/pulp ratio of 1:9 and a moisture content of about
70% of fresh weight. Dewatering was conducted three times for each batch.
Whole berries were fed into the screw press and the pressure was controlled
by applying back-pressure in such a way as to avoid choking. At this stage a
liquid phase (~50% of fresh berry weight) with pulp oil and suspended solids
was separated from a solid fibrous residue. The solid phase was then
resuspended in 25% by weight of hot water and pressed again. This operation
was repeated once more and the three liquid phases were pooled. It was
observed that the weight of the pooled liquid phase was approximately equal
to the fresh weight of berries used in each batch. About 80% of the original
Arimboor et al, JSFA, 86:2345–2353 (2006)
Results & Discussion
148
water content of the berries was separated after the three operations. The
pooled crude juice was then clarified as described above. At 80oC the
suspended solids were found to coagulate and settle at the bottom, leaving a
clear juice above with an orange/red oil layer at the top. High-speed
continuous centrifugation yielded clear orange/red oil (2.7–2.8% of fresh
berry weight) that accounted for 66–70% of the oil present in the soft parts.
The final clear juice obtained was about 75–80% of fresh berry weight (Table
3.16). Results of the compositional analysis of process streams are shown in
Table 3.17. The pulp of the fresh berries used in the process contained 44–46
g/kg of oil on a fresh weight basis. The oil content in the juice (0.3–0.8 g/kg)
and sludge (64–68 g/kg) shows that about 85% of the total oil in the pulp of
berries was extracted with the liquid phase by the high-pressure screw
pressing employed here. The oil content in the fibrous residue (29–32 g/kg)
and sludge together comprised about 25–30% of the oil present in the pulp of
berries. A solvent or supercritical fluid could be employed to recover this
residual oil, but that was not attempted here. The seeds in the fibrous residue
were easily separated after drying followed by air classification. The dried
seeds were ground and their oil was extracted using SC-CO2. The process
streams/products were characterized for their phytochemicals, which are
known to possess biological activity.
Arimboor et al, JSFA, 86:2345–2353 (2006)
Results & Discussion
149
Table 3.16 Results of process development trials of fresh sea buckthorn berries.
Trial
No.
Berries
(kg)
Seed
pulp
ratio
Water
Added
(kg)
Liquid
phase
(kg)
Wet
fibrous
residue
a
(kg)
Wet
solid
sludge
(kg)
Juice
(kg)
Pulp Oil
Qty.
(kg)
Yield on
fresh berries
(%)
Extraction
efficiency
(%)
1
90.0
1:9.3
16.0
89.2
19.0
6.8
73.3
2.51
2.79
68.1
2
35.0
1:9.4
7.5
34.1
8.1
3.8
27.4
0.97
2.77
69.3
3
47.0
1:9.2
9.0
45.3
10.0
6.5
35.1
1.25
2.72
66.5
a Mass includes seeds.
Arimboor et al, JSFA, 86:2345–2353 (2006)
Results & Discussion
150
Table 3.17. Compositional analysis of process streams (%) a.
Tria
l No.
Process
streams Solids Fat Protein Ash
Soluble
sugars Crude
fiber
1
Pulp of
Berries
27.9 ±
0.1 4.6 ± 0.1
1.8 ±
0.2 1.0 ± 0.1
9.5 ±
0.4 2.4 ± 0.1
Juice 13.2 ±
0.4 0.1 ± 0.0
0.5 ±
0.1 0.8 ± 0.0
5.8 ±
0.2 tr
c
Fibrous
Residue b
36.2 ±
0.5 3.0 ± 0.2
5.9 ±
0.3 0.7 ± 0.1
11.8 ±
0.3 9.8 ± 0.7
Sludge 20.2 ±
0.2 6.8 ± 0.4
2.4 ±
0.1 0.8 ± 0.1
4.5 ±
0.4 1.1 ± 0.1
2
Pulp of
Berries
26.2 ±
0.1 4.4 ± 0.1
2.1 ±
0.2 1.0 ± 0.0
9.2 ±
0.5 2.4 ± 0.1
Juice 10.7 ±
0.3 0.0 ± 0.0
0.4 ±
0.0 0.6 ± 0.1
4.9 ±
0.1 tr
c
Fibrous
Residue b
38.7 ±
0.1 3.2 ± 0.1
6.4 ±
0.5 0.6 ± 0.1
11.8 ±
0. 5
12.1 ±
0.5
Sludge 19.1 ±
0.1 6.7 ± 0.2
2.0 ±
0.4 1.0 ± 0.1
4.8 ±
0.2 0.8 ± 0.1
3
Pulp of
Berries
26.9 ±
0.1 4.4 ± 0.1
2.2 ±
0.2 1.0 ± 0.1
9.8 ±
0.4 2.4 ± 0.2
Juice 10.9 ±
0.2 0.0 ± 0.0
0.5 ±
0.2 0.5 ± 0.0
4.9 ±
0.1 tr
c
Fibrous
Residue b
34.9 ±
0.0 2.9 ± 0.2
6.1 ±
0.4 .8 ± 0.1
11.3 ±
0. 4
10.1 ±
0.5
Sludge 19.5 ±
0.1 6.4 ± 0.1
2.1 ±
0.2 1.0 ± 0.0
4.7 ±
0.4 0.6 ± 0.1
a Values are mean ± SD, n=3.
b analyzed and expressed without seed.
c tr ≤ 1g kg
-1
Arimboor et al, JSFA, 86:2345–2353 (2006)
Results & Discussion
151
3.3.2. Chemical Evaluation of the products
Chemical composition of products was evaluated and compared with
that of fresh berries used for the process. Juice, pulp oil and seed oil were
analyzed in detail and retention of bioactive phytochemicals was estimated.
3.3.2.1. Juice.
The raw juice obtained from the berries contained oil droplets and
granules or clumps of insoluble brown solids, which imparted a less
appreciable yellowish brown color to the juice. Clarification followed by
centrifugation removed most of this oil and brown colored solids and resulted
in a clear juice with attractive yellow color. Juice from each batch was
analyzed for its chemical constituents. The final juice contained a relatively
higher amount of solids (10.7–13.2%) in spite of the addition of water, which
could be attributed to high-pressure screw pressing (Table 3.17). The oil
content in the juice was found to be very low (0.03–0.08%) but sufficient to
provide the characteristic flavor of fresh berries (Table 3.17). The presence of
more than 0.1% of oil in juice is a negative quality, since it forms an oily ring
on the top during storage and leads to storage problems such as rancidity
(259). The juice also contained 1–2 mg/kg of carotenoids and about 1 mg/kg
of tocopherols owing to the trace amount of oil. The protein content in the
juice was very low (0.4–0.5%), as most of the protein was found in the
residue (5.9–6.4%) and sludge (2.0–2.4%) (Table 3.16). Total soluble sugars
varied from 4.9 to 5.8% in the juice, accounting for 40–50% of total solids
(Table 3.17). Present reports indicate that glucose and fructose respectively
represent 49.5-62.1% and 37.3-50.4% of total sugars in the juice (147).
Arimboor et al, JSFA, 86:2345–2353 (2006)
Results & Discussion
152
Table 3.18. Polyphenols and flavonoids in the pulp of berries and juice (mg/kg)
a
Composition Pulp of
berries
Juice
Trial 1 Trial 2 Trial 3
Total polyphenols 5646 ± 50 2821 ± 6 2392 ± 15 2757 ± 23
Flavonoids
Quercetin 109 ± 8 81 ± 3 77 ± 4 77 ± 5
Kaempherol 38 ± 2 16 ± 2 12 ± 2 14 ± 2
Isorhamnetin 428 ± 8 303 ± 9 251 ± 15 310 ± 14
Total flavonoids 575 ± 17 400 ± 10 340 ± 19 401 ± 15
a Values are mean ± SD, n=3.
Polyphenols and flavonoids: SB berries used for the process and the juice
obtained were analyzed for their polyphenol content and the results are
presented in Table 3.18. Clear juice contained 2392 – 2821 mg/kg of
polyphenols. 40% of the polyphenols of the berries was found to be extracted
with juice. Flavonoids being an important class polyphenols were analyzed in
detail. HPLC profiling of flavonoid aglycones showed isorhamnetin as the
major flavonoid (428 mg/kg) in the pulp of berries, along with quercetin (109
mg/kg) and kaempherol (38 mg/kg) (Table 3.18). Like other water-soluble
phytochemicals, the juice retained 50–60% of flavonoids of berries. Juice
flavonoid profile included 251–310 mg/kg of isorhamnetin, 77–81 mg/kg of
quercetin and 12–16 mg/kg of kaempherol (Table 3.18). Isorhamnetin
contributed 75% of total flavonoids in berries and juice. Flavonoids
Arimboor et al, JSFA, 86:2345–2353 (2006)
Results & Discussion
153
contributed 10 and 14% of total phenolics in berries and juice respectively.
Polyphenols imparts astringency to fruit juices. PAs, catechins and phenolic
acids were the other major phenolics reported in SB berries (164). The high
amount of flavonoids and other polyphenols present in the juice obtained
enhances this fruit’s nutraceutical value.
Organic acids: The titrable acidity of the final juice was found to be in the
range 3.56 – 3.69% in terms of malic acid, while the pH was between 3.1 and
3.3 (Table 3.19). A wide range (1.9 -5.1%, in terms of malic acid) of titrable
acidity has been reported for SB juice of different origins (147, 260).
Detailed profiling of low-molecular-weight organic acids using HPLC
showed quinic acid (2.80%) and malic acid (1.60%) as the major organic
acids in the pulp of SB berries along with citric acid (0.16%) and traces of
fumaric acid and maleic acid (Table 3.19). The process reported here
extracted about 55–70% of organic acids from the berries into the juice, with
a ratio identical to that of the berries. The juices had 3.0 – 3.6% organic acids
with quinic acid (1.81-1.99%), malic acid (1.17 - 1.48%) and citric acid (0.09
- 0.13%) as major constituents. Sugar acid ratio of the juices ranged from 1.33
to 1.63. A wide range (0.4 -1.9) of sugar acid ratio has been reported for SB
(H. rhamnoides) juices (260). Sugar acid ratio is an important factor in
determining the sweetness or sourness and acceptance of fruit juices. Sugar
acid ratio negatively correlates with the sourness and astringency of fruit juice
(260,261)
The higher amount of quinic acid (1.81–1.99%) in the juice further
highlights the nutraceutical value of SB juice. G-I protective effects of quinic
acid and its derivatives are well established through in-vitro and in-vivo
studies (236). The organic acid profile and the amount of quinic acid can be
used as a chemical marker for evaluation of the quality of commercial SB
juice.
Arimboor et al, JSFA, 86:2345–2353 (2006)
Results & Discussion
154
Table 3.19. pH, titrable acidity, organic acids and vitamin C in the pulp
of berries and juice (mean ± SD, n=3) .
Composition Pulp of
berries
Juice
Trial 1 Trial 2 Trial 3
Total titrable
acids (%) b - 3.56 ± 0.02 3.69 ± 0.04 3.64 ± 0.02
pH - 3.2 3.3 3.1
Organic acids (%)
Quinic acid 2.80 ± 0.07 1.99 ± 0.08 1.85 ± 0.03 1.81 ± 0.02
Malic acid 1.60 ± 0.05 1.48 ± 0.02 1.17 ± 0.02 1.18 ± 0.02
Citric acid 0.16 ± 0.02 0.13 ± 0.01 0.16 ± 0.01 0.09 ± 0.00
Total acids 4.56 ± 0.01 3.60 ± 0.06 3.18 ± 0.05 3.08 ± 0.03
Vitamin C (mg/kg) 2232 ± 42 1840 ± 10 1683 ± 12 1752 ± 17
b Expressed in terms of malic acid.
Vitamin C: SB berries are well known for their extraordinarily high levels of
vitamin C, which is considered as an important quality parameter of SB juice.
The pulp of the Indian berries used in the process contained 2232 mg/kg of
vitamin C (Table 3.19). Approximately 75% of the vitamin C in the pulp of
berries was retained in the juice in this process, resulting in 1683–1840 mg/kg
of vitamin C in the final clear juice. The high vitamin C concentrations make
SB berry highly suitable for the production of nutritious soft drinks. Origin,
maturity and harvesting time of berries and processing techniques and storage
conditions also affect the vitamin C content of the juice
(127,145,147,251,252). Vitamin C content ranging from 290 to 1760 mg/kg
Arimboor et al, JSFA, 86:2345–2353 (2006)
Results & Discussion
155
has been reported for the juices obtained from H rhamnoides berries of
Finnish origin (260)
3.3.2.2 Pulp oil
The pulp oil obtained by the process retained its characteristic flavor in
contrast to the solvent extracted oil. The chemical profile of the pulp oil was
examined in detail.
FA Composition: The FA profile of the pulp oil obtained from the process
was determined using gas chromatography and is summarized in Table 3.20.
The proportions of palmitoleic, palmitic and linoliec acids in pulp oil were
45.6–49.1, 30.9–33.8 and 4.6–5.8% respectively. The uniqueness of SB pulp
oil in terms of the proportion of palmitoleic acid enhances its nutritional
value.
Carotenoids: Total carotenoids in the pulp of berries and pulp oil were
quantified and expressed in terms of β-carotene (Table 3.21). A carotenoid
content of 128 mg/kg was found in the soft parts of berries. Variety, maturity,
climatic conditions and method of oil separation influence the carotenoid
content in oil. Variations in the carotenoid content in pulp oil between batches
could be attributed to the relative proportion of immature berries. The pulp oil
obtained by this process was significantly rich in carotenoids (2450–2810
mg/kg), making SB berries one of the richest natural sources of carotenoids.
Arimboor et al, JSFA, 86:2345–2353 (2006)
Results & Discussion
156
Table: 3.20 FA composition of pulp oil.
Trial No FA (wt %)
16:0 16:1 18:0 18:1 18:2
I 33.8 ± 1.5 46.4 ± 2.0 1.0 ± 0.5 13.4 ± 0.9 5.4 ± 1.4
II 31.2 ± 1.4 49.1 ± 3.1 0.8 ± 0.1 13.9 ± 3.5 4.6 ± 0.6
III 30.9 ± 1.3 45.6 ± 2.6 0.9 ±0.4 16.8 ± 1.7 5.8 ± 0.4
Table 3.21. Carotenoid, tocopherol, tocotrienol and sterol content in the
pulp of berries and pulp oil (mg/kg) a.
Composition Berry Pulp Pulp Oil
Trial 1 Trial 2 Trial 3
Carotenoids 128 ± 2 2810 ± 26 2636 ± 20 2450 ± 14
Tocopherols/tocotrienols
αααα-T b 47 ± 2 987 ± 22 1141 ± 11 1067 ± 35
αααα-T3 c 7 ± 1 182 ± 14 131 ± 8 94 ± 6
ββββ-T b 1 ± 0 28 ± 4 22 ± 2 21 ± 2
γγγγ-T b 5 ± 1 91 ± 2 134 ± 10 109 ± 12
γγγγ-T3 c +δδδδ-T
b 6 ± 1 107 ± 8 154 ± 11 134 ± 6
δδδδ-T3 c 1 ± 0 14 ± 1 17 ± 1 15 ± 1
Total 67 ± 2 1409 ± 28 1599 ± 14 1439 ± 48
Sterols
ββββ-sitosterol 180 ± 8 3200 ± 49 3221 ± 36 3034 ± 48
Stigmasterol 80 ± 8 1203 ± 24 1058 ± 35 1062 ± 39
Total 260 ± 15 4403 ± 35 4279 ± 30 4096 ± 57
a Values are mean ± SD, n=3.
b T : Tocopherol;
c T3 : Tocotrienol
16:0; palmitic acid, 16:1; palmitoleic acid, 18:0; stearic acid, 18:1; oleic acid, 18:2;
linoleic acid
Arimboor et al, JSFA, 86:2345–2353 (2006)
Results & Discussion
157
Tocopherols and tocotrienols: The pulp of the berries used in the process
contained 67 mg/kg of tocopherols and tocotrienols (Table 3.21). The pulp
oil obtained by the process had 1409–1599 mg/kg of tocopherols and
tocotrienols. α-Tocopherol, the anomer with maximum vitamin E activity
among tocols, constituted 69–73% of total tocols in pulp oil. α-Tocotrienol, γ
-tocopherol, δ-tocopherol and γ -tocotrienol were other tocols identified in
pulp oil. Traces of β-tocopherol and δ-tocotrienol were also detected in pulp
oil.
Sterols : β-Sitosterol (180 mg/kg) and stigmasterol (80mg/kg) were found to
be the major sterols in the pulp of berries, with unidentified peaks indicating
the presence also of minor compounds (Table 3.21). Trace amounts of
campesterol, a major sterol in seed oil, were also detected in pulp oil. Pulp oil
obtained by the present process showed a similar sterol profile of berries, with
3034–3200 mg/kg of β-sitosterol and 1058–1203 mg/kg of stigmasterol.
3.3.2.3. Seed oil
Seeds from Indian grown berries have not been studied in detail for
their nutraceutical values. As part of the integrated approach to processing SB
berries, a comparative evaluation of seed oils obtained by hexane and SC-CO2
extraction was made with the seeds obtained from the process reported here.
The yield of hexane-extracted oil was slightly higher (6.1% by wt. of seed)
than that of SC-CO2-extracted oil (5.2% by weight of seed). All
phytochemical constituents (carotenoids, tocopherols/tocotrienols and sterols)
were more abundant in the oil extracted with SC-CO2 than in the oil extracted
with hexane. The lower content of phytochemicals in hexane-extracted oil
could be due to non-oil components such as wax and gum being extracted
with hexane, resulting in a higher extract yield with a lower concentration of
phytochemicals (Table 3.23). The SC-CO2 extraction conditions employed
here (60◦ C temperature, 4.5 × 107 Pa pressure and 60 g/min gas flow rate for
Arimboor et al, JSFA, 86:2345–2353 (2006)
Results & Discussion
158
3 h) were able to extract seed oil with micronutrients as effectively as or
better than hexane.
Seed oil differed significantly from pulp oil in its chemical
characteristics. While palmitoleic acid (45–49%) was the most predominant
FA in pulp oil, its content in seed oil was as low as 2.1-3.6% (Table 3.22).
The major FAs in seed oil were palmitic (17-18%), oleic (29-30%), linoleic
(26-28%) and ALA (16-19%). Seed oil with more than 16% ALA otherwise
called omega-3-FA is rare among vegetable oils. The biological properties of
ALA are well documented. SB seed oil as a vegetable oil with high levels of
ALA, therefore had nutraceutical value comparable with fish oil. The higher
level of carotenoids in pulp oil (2450-2810 mg/kg) than in seed oil (~400
mg/kg) was another major difference. As reported above, the total tocol
content in seed oil (1012 mg/kg) was also lower than that in pulp oil (1409-
1599 mg/kg) (Tables 3.21 & 3.23). The γ -tocopherol content in pulp oil was
lower than that in seed oil. The results for tocols indicate that, owing to its
higher total tocol content with a high proportion of α-tocopherol, pulp oil had
higher vitamin E activity than seed oil. The total sterol content in seed oil
(16887 mg/kg) was about four times that in pulp oil (Tables 3.20 & 3.22). β-
Sitosterol (13774 mg/kg) was the major sterol in seed oil. Seed oil also
contained significant amounts of campesterol (1878 mg/kg) and stigmasterol
(1235 mg/kg).
Arimboor et al, JSFA, 86:2345–2353 (2006)
Results & Discussion
159
Fatty acids
16:0 16:1 18:0 18:1 18:2 18:3
Hexane
extracted 17.9±1.2 2.1±0.3 4.1±0.6 29.8±1.0 26.1±0.8 18.8±1.2
SC- CO2
Extracted 17.2±1.2 3.6±0.2 3.8±0.4 30.0±1.2 27.7±0.8 16.7±0.7
Table 3.23. Yield, Carotenoid, tocopherol, tocotrienol and sterol content
in the hexane and SC- CO2 extracted seed oils (mg/kg) (Mean ± SD).
Composition Seed Oil
Hexane extracted SC-CO2 extracted
Carotenoids 387 ± 6 403 ± 9
Tocopherols/tocotrienols
αααα-T b 520 ± 14 587 ± 12
αααα-T3 c 11 ± 1 35 ± 4
ββββ-T b 19 ± 1 21 ± 2
γγγγ-T b 306 ± 12 315 ± 14
γγγγ-T3 c +δδδδ-T
b 46 ± 3 39 ± 4
δδδδ-T3 c 10 ± 1 15 ± 1
Total 912 ± 20 1012 ± 26
Sterols
ββββ-sitosterol 13305 ± 76 13774 ± 112
Stigmasterol 1125 ± 64 1235 ± 57
Campesterol 1770 ± 29 1878 ± 57
Total 16200 ± 150 16887 ± 87
a Values are mean ± standard error, n=3. b T : Tocopherol; c T3 : Tocotrienol
Table.3.22 FA composition (wt. %) of hexane and supercritical CO2
extracted seed oils (mean ±±±± SD)
Arimboor et al, JSFA, 86:2345–2353 (2006)
16:0; palmitic acid, 16:1; palmitoleic acid, 18:0; stearic acid, 18:1; oleic acid, 18:2; linoleic acid
Results & Discussion
160
The reports related to SB processing are rather limited (129,177, 179)
and show a fragmented approach. Beveridge et al; discussed the available
processes in a recent review (129). According to them, the main products
from SB berries are seed oil, yellow pigment and juice. Pulp oil, though very
important in terms of its quantity and phytochemical profile as compared with
seed oil, was not given adequate attention by the authors, perhaps owing to
the limited data being available on pulp oil recovery. The general approach to
processing SB involved dewatering and juice clarification as the critical
process steps (129,262,263). Details of material balance, efficiency of
extraction, product yield, commercial feasibility, etc. have not been discussed
in quantitative terms in the published reports. Hence comparison of the data
from our processing of Indian SB berries is difficult. Our approach has been
to integrate process and products and maximize the yield of juice, pulp oil and
seed oil from fresh berries of SB grown in the Indian Himalayas, hitherto not
attempted. Most of the unit operations employed here are similar to those
reported previously. A high yield of pulp oil and juice by employing a
combination of high-pressure dewatering screw pressing and continuous
centrifugation was achieved in this process. Approximately 80% of the water
and oil and 60% of the solids in the pulp were extracted to the liquid phase
using the high-pressure screw press with a compression ratio of about 10. The
continuous high-speed centrifugation employed here led to efficient
separation of the liquid phase into oil free clear juice, pulp oil and solid
sludge. The yield of clear juice at 75–80% of berry weight with 10–13%
soluble solids appeared to be high owing to effective pressing. Further, the
juice obtained here also retained more than 60% of organic acids, 70% of
vitamin C, 40% of polyphenols and 50% of flavonoids present in the pulp of
berries. The sugar acid ratio of the juices was in higher end of the range
reported in literature. It could be seen that, even after the addition of water to
facilitate dewatering and consequent dilution of juice, the concentration of
total solids and bioactives remained fairly high in the final juice, which could
Arimboor et al, JSFA, 86:2345–2353 (2006)
Results & Discussion
161
be attributed to the extraction efficiency. Beveridge et al; reported the effect
of processing on the composition of SB juice (129). The authors demonstrated
the use of conventional rack and cloth pressing in batch mode. The juice was
treated with pectinase, followed by batch centrifugation for juice clarification,
and the clarified juice was examined for vitamin C, organic acids and sugars.
However, the authors did not discuss the process efficiency. The vitamin C
and organic acid contents of the juice described by them are similar to those
of the juice from Himalayan berries reported here. Considering the high levels
of total solids and other phytochemical constituents, the final juice obtained
here could be further diluted in the ratio 1:2 to formulate a ready-to drink
health beverage rich in bioactive phytochemicals.
Pulp oil is another valuable co-product of this process, having an
extremely high content of carotenoids, tocopherols and sterols with
characteristic fatty acid 16:1. The use of vegetable oils as solvents for the
extraction of pulp and seed oils has also been reported, but the efficiency of
recovery was found to be poor. The present process was able to separate up to
70% of the pulp oil without using organic solvents, so maximum retention of
bioactives was achieved. An important advantage of this process was that
seeds could be recovered without damage in spite of the high-pressure
dewatering of berries. The feasibility of extraction of high-quality seed oil
using SC-CO2 was also demonstrated.
The process reported here, for fresh SB berries grown at high altitudes
in the Himalayas, constitutes an integrated approach to yield products with
high efficiency and quality for nutraceutical applications. SB berries are
highly perishable and have to be processed within hours of harvesting to
obtain quality products. This green integrated approach would also be suitable
for ecologically fragile SB growing areas. Even though SB berries from the
Arimboor et al, JSFA, 86:2345–2353 (2006)
Results & Discussion
162
Himalayas have not been utilized on a commercial scale, the potential appears
to be high following the process reported here.
Results & Discussion
163
3.4. In Vitro AO Activity Evaluation of Sea
Buckthorn (H. rhamnoides) Berries
3.4.1 In Vitro AO Activities of H. rhamnoides Berry Extracts
A wide spectrum of biological activities has been attributed to SB
berries. More than 300 SB medicinal preparations are mentioned in literature
and several preparations have been clinically evaluated to treat radiation
damage, burns, oral inflammation and gastric ulcers (191). Berries and berry
products are also being used in nutraceutical and cosmaceutical applications.
Apart from reports on applications and physiological properties of SB, reports
describing bioactivities of SB berries in relation to their phytochemical
compositions are limited. This part of the present study was aimed at the
evaluation of AO properties of SB berries belonging to H. rhamnoides and
chemical profiling of active fractions.
In plant products, biological activities are often confined to certain
parts and fractions. Activity guided fractionation approach was therefore
applied here. Hexane, ethyl acetate, methanol and water extracts of SB berry
anatomical parts (pulp, seed coat and kernel) were prepared and subjected to
AO capacity evaluation. TEAC, DPPH, superoxide and hydroxyl radical
scavenging, Fe(III) reduction and Fe(II) chelation and XO inhibition
capacities of berry extracts were studied using in-vitro model assay systems.
The kernel methanol extract with significant yield and high AO capacity was
further fractionated between hexane, ethyl acetate, n-butanol and water. Each
fraction was evaluated for their AO capacity and active fractions were
subjected to chemical evaluation. Activity guided fractionation scheme used
for the preparation of berry samples is given in Figure 2.2
Results & Discussion
164
Table 3.24. Extraction efficiency in terms of yield of extract of different
solvents on sequential extraction.
The extractability of berry parts as affected by different solvents is
summarized in Table. 3.24. Methanol and water as solvents showed high
extractability of berry parts. 94.8% of the total dry matter in the pulp could be
extracted with the solvents whereas total solubles in the seed coat and kernel
were 18.5 and 41.5% respectively. The low soluble matter content of seed
coat might be attributed to its high proportion of high molecular weight
structural components.
Berry parts Solvents Extraction Efficiency
(in dry wt. of samples) (%)
Pulp
Hexane (PHE) 14.25
Ethyl Acetate (PEE) 19.8
Methanol (PME) 35.8
Water (PWE) 24.9
Total 94.8
Seed Coat
Hexane (SCHE) 2.3
EtOAc (SCEE) 0.2
Methanol (SCME) 9.2
Water (SCWE) 6.8
Total 18.5
Kernel
Hexane (KHE) 7.2
Ethyl Acetate (KEE) 1.5
Methanol (KME) 13.9
Water (KWE) 18.9
Total 41.5
Results & Discussion
165
Total phenolics by F-C Method
Phenolic compounds are one of the major classes of phytochemicals
with significant AO activities. The extracts were evaluated for their total
phenolics content by using F-C reagent. F-C reagent assay is also considered
as a rough method for AO capacity evaluation (264). Seed coat methanolic
(SCME) and water (SCWE) extracts contained high amounts of phenolics
(32.9 and 24.3 respectively). Phenolic concentration in kernel extracts (KME
= 22.6%, KWE = 16.3%) was also high. Pulp methanol (PME) and water
(PWE) extracts respectively had 6.8 and 4.4% phenolics.
3.4.1.1. DPPH radical scavenging activity
DPPH radical (DPPH●) is a commercially available stable free radical
(Figure 1.6). DPPH● can accept an electron or hydrogen radical to become a
stable diamagnetic molecule. Both hydrogen atom transfer (HAT) and single
electron transfer (SET) mechanisms have been proposed for the quenching of
DPPH● by AOs (265). The quenching of DPPH
● can be monitored by
tracking the decrease in its absorbance at 517nm. Compounds, which can
donate electrons or H●
to quench DPPH●, are considered to have capacity to
scavenge free radicals. DPPH● is widely used as a substrate for the high
through put screening of free radical scavengers.
The reaction curves for the DPPH● scavenging of berry extracts are
given in Figure 3.15 and the IC50 values are summarized in Table 3.24. The
radical scavenging capacity of extracts/compounds was compared in terms of
IC50 values. IC50 value denotes the amount of extract/compounds required to
scavenge 50% of DPPH● in the reaction system (Figure 3.15). In the present
study it was shown that methanol and water extracts of pulp and seed
possessed high DPPH● scavenging efficacy. The results showed that the
SCME (IC50 = 3.0 µg), SCWE (IC50 = 6.6 µg), KME (IC50 = 6.7 µg) and KWE
(IC50 = 19.2 µg) had significantly higher DPPH● scavenging activity. Pulp
Results & Discussion
166
methanol and water extracts respectively showed IC50 values of 654.5 and
796.3 µg. The radical scavenging activities of kernel and seed coat extracts
were comparable with that of standard compounds like gallic acid (IC50 = 1.6
µg), and catechin (IC50 = 5.2 µg). But the yield of SCME was very low as
compared to that of KME and PME (Table 3.24). There are some recent
reports on the high DPPH● radical scavenging activity of SB whole seed
extracts (128, 168). The results of the present study are in accordance with
these reports.
3.4.1.2. Trolox Equivalent Antioxidant Capacity (TEAC).
ABTS cation radical is an important and popular probe used for the
evaluation of AO capacity of biological fluids and phytochemicals. The
method is used to monitor the decay of the radical cation ABTS, produced by
the oxidation of 2, 2’-azinobis(3-ethylbenzothiazoline-6-sulphonate) (Figure
3.14) in presence of AOs. ABTS●+
has a strong absorption at 700-750 nm. H●
donor molecules quantitatively convert ABTS●+
to colorless ABTS molecule.
A mixed type mechanism (HAT and SET) has been proposed for the
scavenging of ABTS●+
by AOs (265). Trolox, an important H● donor is used
as a reference in ABTS assay. The AO capacities of the compounds are
represented as trolox equivalent antioxidant capacity (TEAC). TEAC of 1 mg
of berry extracts is given in Table 3.25. SCME had the highest TEAC value
(8.0 nM), followed by KME (5.8 nM), SCWE (5.6 nM) and KWE (1.1 nM).
ABTS●+
scavenging activity of the berry extracts was in correlation with their
phenolic content. Extracts with high phenolic content had high TEAC values.
Gallic acid and catechin showed TEAC values 12.9 and 9.2 nM respectively.
Due to lack of reports on the TEAC of SB berries discussion cannot be
extended to previous works in this regard.
Results & Discussion
167
Figure 3.14. ABTS
S
EtN
N N
NEt
SSO3H
HO3S
3.4.1.3 Fe(III) reducing power
Fe(III) reduction assay is under the class of SET mediated AO assays.
In this assay the presence of AOs (reductants) would bring about the
reduction of the [Fe(CN)6]3-
to [Fe(CN)6]4-
. Amount of Fe2+
complex can then
be monitored by measuring the formation of Perl’s Prussian blue at 700 nm
with increased absorbance indicating higher reducing power.
Figure 3.16 shows the dose response curves for the reducing powers of
the SBT extracts and reference compounds (gallic acid, trolox and catechin).
It was found that the reducing power of all the extracts was in linear
correlation with their concentration in the experimental range. The Fe(III)
reducing power of 100 µg of samples is expressed as ascorbic acid
equivalents (AAE) in Table 3.25. The reducing power values of 100 µg of
KME, SCME, KWE and SCWE were 21.3, 17.9, 17.0 and 13.2 µg
equivalents of ascorbic acid respectively. Pulp extracts showed very low
reducing power. The reducing power of KME and SCME extracts were
comparable with that of catechin and trolox. Gallic acid showed the highest
reducing power (125 µg AAE) among the reference compounds used here,
followed by catechin (58.4 µg) and trolox (29.6 µg). These results reveal that
SB berry extracts, especially seed extracts can act as electron donors and
could react with free radicals, more efficiently converting them to stable
products, and terminating the radical chain reaction.
Results & Discussion
168
3.4.1.4. Fe(II) chelation capacity
Metals especially Cu and Fe catalyze the generation of free radicals
from neutral molecules and subsequently trigger free radical reactions. Cu+ or
Fe2+
mediated formations of hydroxyl radicals from hydrogen peroxide
(Fenton’s reactions) are typical examples. Therefore the compounds with the
ability to chelate heavy metals and thus keep them unavailable for metal
mediated free radical reactions are also considered as AOs. The metal
chelation capacity of the berry extracts was evaluated using Fe(II)/ferrozine
system, in which Fe2+
ions were incubated with the berry extracts/reference
compounds and the free and loosely bound Fe2+
ions were estimated using
ferrozine. The % of Fe(II) chelated by 1 mg of extracts/reference compounds
are summarized in Table 3.25. Highest chelation capacity was shown by
KME (12.4%) followed by KWE (8.4%), SCME (6.9%) and SCWE (4.8%).
The reference compound EDTA chelated 89% of Fe2+
in the reaction systems
under the experimental conditions. SB berry and its fractions had moderate
metal chelation capacity.
3.4.1.5. Hydroxyl radical scavenging activity
Among the oxygen radicals, hydroxyl radical is the most reactive and
induces severe damage to biomolecules. The scavenging effect of berry
extracts against hydroxyl radical was therefore investigated using the
Fenton’s reaction. Only SB seed extracts showed hydroxyl radical scavenging
activity and the activity increased with increasing concentration. From the
IC50 values of the extracts (Table 3.25), it was revealed that KME (58 µg) had
the highest scavenging activity followed by SCME (92 µg), KWE (109 µg)
and SCWE (176 µg). Among the standard AO compounds tested, gallic acid,
the most effective scavenger of DPPH and ABTS radicals, was less effective
in neutralizing hydroxyl radical under the in vitro assay conditions. Catechin
and trolox respectively had the IC50 values 125 and 141 µg.
Results & Discussion
169
3.4.1.6 Superoxide anion radical scavenging (SOS) activity:
Superoxide radical (O2●▬-
) is one of the harmful free radical found in
biological systems. It attacks cellular components as a precursor of the more
reactive oxygen radicals having the potential of reacting with biological
macromolecules and thereby inducing tissue damage (24). In biological
system, its toxic role can be eliminated by superoxide dismutase (SOD).
There is an increasing interest in identifying active compounds from plant
materials that can exhibit activity similar to SOD. SOS activity of SB berry
extracts/reference compounds was measured using the xanthine-XO system.
Xanthine-XO generated superoxide radicals reduce NBT to formazan blue
(500 nm). Decreased formation of formazan blue is considered as the
indication of SOS activity of samples. But in fact the reduction in blue
coloration of reaction system may also be due to the reduction in the
generation of superoxide by the inhibition of XO enzyme activity by samples.
Therefore the inhibition of XO by the extracts/reference compounds was also
studied and discussed in the next section.
The SOS activities of SB berry extracts are compared as IC50 values
and the results are summarized in Table 3.25. Methanol and water extracts of
seed coat and kernel exhibited SOS activity comparable to that of gallic acid
and catechin in a dose-dependent manner. KME (69 µg) showed the highest
activity among the tested samples followed by SCME (79 µg), KWE (197 µg)
and SCWE (128 µg). SB pulp extracts didn’t show significant scavenging
activity. The results reveal that SB seed and seed coat extracts are potent
scavengers of superoxide radicals or inhibitors of XO activity, or both.
Results & Discussion
170
3.4.1.7. Xanthine oxidase (XO) inhibition capacity.
Xanthine to uric acid conversion by XO is an important step in purine
metabolism. This conversion is associated with the generation of superoxide
radical anion. Hyperactivity of XO or the reduced removal of uric acid from
the body leads to hyperurecimia, gout, arthritis and other inflammatory
diseases. The compounds which are able to inhibit the XO activity are used to
control the uric acid deposition in hyperurecemic patients. Allopurinol, a
potent inhibitor of XO is used as a drug against hyperurecemic diseases like
gout. This compound has reported to have many side effects. Therefore the
search for potential XO inhibitory compounds from natural sources attracts
special interest.
The SOS capacity of AOs evaluated using xanthine/XO NBT assay
depends on both their ability to inhibit XO enzyme activity and to scavenge
superoxide radicals. Therefore berry extracts were also evaluated for their XO
inhibitory efficacy. The XO inhibitory efficacies of the extracts expressed as
IC50 values are summarized in Table.3.25. Highest activity was shown by
KME (121 µg), followed by SCME (193 µg), KWE (203 µg) and SCWE (291
µg) (Table 3.25). No significant XO inhibition was shown by pulp extracts.
Allopurinol and catechin respectively showed IC50 values 14 and 39 µg under
the experimental conditions. These results showed that SB seeds are good
source of XO inhibiting compounds. This is the first report on the XO
inhibitory capacity of SB berry extracts.
Results & Discussion
171
Table-3.25: TPC and in vitro antioxidant capacities of SB berry extracts and reference compounds (m
ean±SD. n=5).
SB extracts/
Antioxidant compounds
Total
phenolics
(GAE)
%
DPPH*
Scavenging
Activity
IC50 (
µg)
TEAC
(nM
)
Fe(III)
reducing
capacity/100 µ
g
[(AAE)
(µg)]
Fe(II)
Chelation
capacity
%/m
g
Hydroxyl
radical
scavenging
IC50 (
µg)
Superoxide
radical
scavenging
IC50 (
µg)
Xanthine
Oxidase
inhibition
IC50 (µ
g)
Pulp
Methanol (PME)
6.8
± 0
.3
654.5
± 2
9.6
N
.S
N.S
N
.S
N.S
95
2 ±
36
N.S
Water (PWE)
4.4
± 0
.1
796.3
± 2
1.9
N
.S
N.S
N
.S
N.S
16
54 ±
89
N
.S
Kernel
Methanol (K
ME)
22.6
±
2.1
6.7
± 0
.6
5.8
± 0
.3
21.3
± 0
.4
12.4
± 1
.9
58 ±
6
69 ±
11
122 ±
11
Water (KWE)
16.3
± 2
.4
19.2
±
0.9
1.1
± 0
.2
17.0
± 0
.2
8.4
± 1
.2
10
9 ±
8
19
7 ±
29
203 ±
13
Seed
Coat
Methanol
(SC
ME
) 32.9
± 1
.9
3.0
±0.3
8.0
± 0
.6
17.9
± 0
.6
6.9
± 1
.2
92 ±
12
79 ±
17
193 ±
21
Water (SCW
E)
24.3
± 3
.2
6.6
± 0
.8
5.6
± 0
.6
13.2
± 1
.1
4.8
± 0
.8
17
6 ±
22
12
8 ±
12
291 ±
18
Gallic acid
- 1.6
± 0
.0
12.9
± 0
.9
125.4
± 0
.1
- N
.S
35
± 1
2
112 ±
09
Catechin
- 5.2
± 0
.0
9.2
± 0
.4
58.4
± 0
.2
- 12
5 ±
4
18 ±
4
39
Trolox
-
- 29.6
± 0
.3
- 14
1 ±
18
34
4 ±
10
-
EDTA
- -
- -
89.3
± 3
.4
- -
-
Allopurinol
- -
- -
- -
- 14 ±
2
GA
E :
Gal
lic a
cid
eq
uiv
ale
nts
, A
AE
: A
scorb
ic a
cid e
quiv
ale
nts
, N
.S :
Not
sign
ific
ant
Results & Discussion
172
Figure 3.15. DPPH radical scavenging efficacies of H . rhamnoides berry
extracts and reference compounds (mean + S D (n = 5)).
0 2 4 6 8 10 12 14 16
0
20
40
60
80
100% RSA
Amount of Extracts/Compounds (micro gm)/mL
GA
Cat
KME
SCME
SCWE
0 200 400 600 800 1000
PME
GA : Gallic acid, Cat : catechin, KME : kernel methanol extract, KWE : kernel
water extract, SCME : seed coat methanol extract, SCWE : seed coat water extract,
PME : pulp methanol extract.
Results & Discussion
173
Figure 3.16. Fe(III) reduction capacities of H rhamnoides berry extracts
and reference compounds.
0 20 40 60 80 100 120 140 160 180 200 220 240 2600.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
Vit C
GA
Cat
Tx
KME
KWE
SCME
SCWE
OD
at 700 n
m
Conc. in microgram
Vit C: vitamin C, GA : Gallic acid, Cat : catechin, KME : kernel methanol
extract, KWE : kernel water extract, SCME : seed coat methanol extract,
SCWE : seed coat water extract.
AAE
Results & Discussion
174
3.4.2. AO Capacity Evaluation of KME Fractions
Methanol extracts of seed coat and kernel were found to have high AO
capacity and phenolic content. But the yield of seed coat extracts (~ 0.25% of
dry mass of berries) was very low as compared to that of kernel extracts (~
2.3% of dry mass of berries). Therefore kernel methanol extract (KME) was
selected for further studies. KME thus was fractionated between hexane
(KMHF), ethyl acetate (KMEF), butanol (KMBF) and water (KMWF). The
yield of KME fractions is summarized in Table.3.26. Major portion of KME
was resided with water fraction (50.5%). 6.9, 18.6 and 11.9% of KME were
respectively resided into KMHF, KMEF and KMBF.
KME fractions thus prepared were subjected to in-vitro AO capacity
studies and active fractions were evaluated for their chemical composition.
3.4.2.1 Total phenolics
The amount of phenolics of KME fractions as obtained by F-C reagent
method is given in Table 3.26. Approximately 80 % of phenolics in the KME
were recovered among the fractions. KMEF (35.7%) had the highest
concentration of phenolics followed by KMWF (20.8%), and KMBF (18.4%).
KMHF (2.9%) had the lowest phenolic concentration. Phenolic content in
KMEF was enriched with respect to KME. Approximately 45% of total
phenolics in the extract were found to be retained with KMWF fraction and
33% with KMEF.
3.4.2.2. In vitro AO capacities of KME fractions
AO capacity of KME fractions was evaluated by using in-vitro model
assay systems and the results are tabulated in Table 3.26. KMEF (IC50 = 4.9
µg) and KMWF (IC50 = 4.1 µg) had higher DPPH● scavenging activity than
the parent extract KME (IC50=6.7 µg). Their DPPH● scavenging activity were
comparable with that of standard compounds like gallic acid (IC50 = 1.6 µg),
Results & Discussion
175
and catechin (IC50 = 5.2 µg) (Table 3.25 & 3.26). Other fractions had lower
activity than KME. The correlation between DPPH● scavenging capacity and
phenolic concentration of KME fractions was very poor. The KMEF with the
highest phenolic content (35.7%) had scavenging capacity lower than that of
KMWF with moderate phenolic content (20.8%). This indicated that
phenolics in the KMWF were more potent as DPPH●
scavengers than those of
KMEF.
TEAC for 1 mg of fractions of KME is given in Table 3.26. TEAC of
KME (5.8 nM) was found to be enriched in KMEF (8.0 nM) and KMWF (8.5
nM) fractions. KMHF (0.1 nM) and KMBF (1.1 nM) were having lower
activity than KME. TEAC values of extracts were also not in correlation with
their phenolic content.
Fe(III) reducing power of KME fractions are shown in Table 3.26.
KMWF (32.0 µg AAE) had the highest Fe(III) reducing power among the
fractions. KMEF and KMBF respectively showed 26.9 and 19.3 µg AAE
reduction power. Fe(II) chelating capacity of the KME was also enriched in
its ethyl acetate and water fractions. 1 mg of KMEF and KMWF respectively
chelated 16.8 and 14.6% of Fe(II) ions present in the reaction system.
Chelation capacity of the extract was in line with their phenolic content
(Table 3.26).
Hydroxyl radical scavenging capacity of the KME fractions was found
to be distributed among the fractions. KMEF (IC50 = 39 µg) and KMWF (IC50
= 48 µg ) showed slightly higher activity than KME ( IC50 = 58 µg). KMBF
(IC50 = 68 µg) had slightly lower activity than KME Table 3.26. Hydroxyl
scavenging capacity of fractions showed a positive correlation with their
phenolic content.
Results & Discussion
176
SOS activity of KME fractions are presented in Table 3.26. KMWF
(IC50 = 54 µg) showed the highest activity among the fractions followed by
KMEF (IC50 = 80 µg) and KMBF (IC50 = 145 µg) fractions. XO inhibitory
efficacy evaluation of KME fractions showed that the activity was
concentrated in KMEF (IC50 = 78.2 µg). KMWF ((IC50 = 109 µg) also showed
higher activity than KME ((IC50 = 122 µg).
DPPH● scavenging, TEAC, Fe(III) reducing and SOS capacities of
KME fractions failed to correlate with their phenolic content. This could be
due to the distinct characteristics of its phenolic constituents that warranted
detailed chemical characterization of these fractions.
Results & Discussion
177
Table 3.26: Yield, phenolic content and in vitro AO capacities of KME fractions and reference compounds (m
ean±SD.
GA
E :
Gall
ic a
cid
equiv
alent,
AA
E :
Asc
orb
ic a
cid
eq
uiv
ale
nt.
SKME Fractions/
Reference
compounds
Yield
(%)
Total
Phenolics
(%)
(GA
E)
DPPH*
Scavenging
Activity.
IC50 µ
g/m
L
TEAC
(nM)
Fe(III)
Reducing
Capacity/
100 µ
g
[(AAE)
µg]
% of
Fe (II)
Chelation
/mg
Hydroxyl
radical
scavengin
g
Capacity.
Superoxi
de radical
ion
scavengin
g
xanthine
oxidase
inhibition
(µg)
Methanol Extract
(KME)
- 22
.6 ±
1.1
6.7
±
0.6
5.8
± 0
.2
21.3
± 0
.4
12.4
± 0
.4
58 ±
6
69 ±
11
122 ±
9.2
Hexane (KMHF)
6.9
2.9
± 0
.6
18.9
± 0
.4
0.1
± 0
.0
0.6
± 0
.0
NS
N
.S
854 ±
14
NS
Ethyl acetate
(KMEF)
18.6
35
.7 ±
1.8
4.9
± 0
.6
8.0
± 0
.2
26
.9 ±
0.2
16.8
± 1
.1
39 ±
6
80 ±
6
78 ±
10.1
Butanol (K
MBF)
11.9
18
.4 ±
2.2
12.3
± 0
.4
1.1
± 0
.4
19.3
± 0
.4
8.5
± 1
.2
68 ±
4
14
5 ±
12
451 ±
12.6
Water (KMWF)
50.5
20
.8 ±
1.9
4.1
± 0
.5
8.5
± 0
.6
32.0
± 1
.4
14.6
± 1
.5
48 ±
8
54 ±
11
109 ±
9.8
Results & Discussion
178
3.4.3. Chemical profiling of active fractions
Both ethyl acetate and water fractions of KME found to have
considerably high AO capacity than the parent KME. This showed that major
portion of the compounds responsible for the AO activity of KME was
concentrated in KMEF and KMWF. The KMEF and KMWF contributed
approximately 78% of the phenolics in the KME. Therefore both KMEF and
KMWF were subjected to further fractionation and detailed chemical analysis.
Phenolic compounds have been reported as the major AO compounds in
plants. A large number of reports on the direct correlation between in-vitro
AO activities and phenolic content of plant extracts are available (128,168).
Therefore phenolics as major AO compounds in this extract were subjected to
detailed composition analysis.
3.4.3.1 Chemical profiling of KMEF.
KMEF was subjected to detailed chemical analysis. Phenolics
contributed considerable amount (35.7%) of solids in KMEF. Total flavonols
estimation using AlCl3 method showed a flavonol concentration of 30.4% in
KMEF which contributed 85% of phenolics. This indicated that flavonols are
the major phenolics in KMEF. Therefore detailed profiling of flavonoids in
KMEF was attempted. Flavonoids are usually found as their free and
glycosidic forms in plant kingdom. The variations in the number and nature of
glycosidic bonds and carbohydrate moiety result in large diversity of
flavonoid glycosides. Structural resemblance and close elution properties with
common chromatographic conditions make the isolation and identification of
the flavonoid glycosides a laborious task. Recently, the development of
hyphenated techniques such as LC-MS and LC-NMR considerably reduces
the time and labor required for the flavonoid glycoside analysis. In this study
the AO active KMEF was subjected to LC-MS/MS profiling. LC-MS is a
hyphenated technique in which the resolution of compounds takes place in
liquid chromatograph and the fractions are subsequently subjected to mass
Results & Discussion
179
analysis. Mass spectra along with HPLC retention data provides an effective
tool for qualitative and quantitative analysis of phytochemicals and
biomolecules.
3.4.3.1a HPLC-DAD-ESI-APCI-MS profiling of flavonoid glycosides in
KMEF. HPLC equipped with both DAD and MS/MS as detectors was used
for the flavonoid analysis. Under the optimized chromatographic conditions,
KMEF resulted more than 20 peaks in chromatogram at 360nm (Figure 3.17).
Major flavonoid glycosides were identified by examining the MS and UV
spectra and by comparing with the available literature on flavonoids. Reports
on the flavonoid glycosides in SB juice (164), pomace (266) and berry pulp
(166) provide valuable indications for their structural characterization. The
identified flavonol glycosides in the fraction were grouped into four viz.
mono glucosides, mono rutinosides, 3, 7, diglycosides and 3- sophroside-7-
rhamnosides (Figure 3.18).
Quercetin (Peak No.9), kaempherol (Peak No.10) and isorhamnetin
(Peak No.11) were identified by comparing with reference samples (Figure
3.16). Quercetin, kaempherol and isorhamnetin were characterized by their
[M-H]▬
and [M-15]▬
mass peaks (Table 3.27). Calibration curves using these
reference compounds were plotted and used for the quantification of
corresponding glycosides.
Isorhamnetin-3-O-glucoside (Peak No. 8) was identified by comparing
the characteristic mass spectral peaks obtained at m/z 477 ([M-H] ▬
) and m/z
315 ([(M-H)-162]▬
) with previous reports (266). Quercetin-3-O-rutinoside
(Peak No.6) was identified by the comparison with authentic reference
substance. MS-MS fragmentation of molecular ion at m/z 609 ([M-H]▬
)
produced the pseudo molecular ion at m/z 301 ([(M-H)-208]▬
). A similar
fragmentation pattern was observed for isorhamnetin-3-O-rutinoside (Peak
Results & Discussion
180
No.7) [M-H]▬
at m/z 623 and [(M-H)-208]▬
at m/z 315. A complete loss of
disaccharide, rutinose without the formation of a fragment [(M-H)-146]▬
from flavonol rutinosides has been reported (266) (Figure 3.17 & 3.18).
In contrast to quercetin-3-O-rutinoside, compound 4 (Peak No. 4)
resulted in abundant daughter ion at m/z 463 ([(M-H)-146]▬
) corresponding
to a loss of rhamnose. The daughter ion at m/z 447 [M-H- 162] ▬
resulting
from the loss of glucose had lower intensity than that at m/z 463. Llorach et
al; demonstrated the higher sensitivity glycosidic linkage at C-7 position than
C-3 position towards collision-induced fragmentation (267). Based on these
assumptions compound 4 was assumed to be quercetin-3-O-glucoside-7-O-
rhamnoside. Compound 5 with similar pattern of mass spectra was identified
as isorhamnetin-3-O-glucoside-7-O-rhamnoside (Figure 3.17 & 3.18).
.
The compound 1 showed a [M-H]▬
at 771, which on MS-MS
fragmentation resulted in a mass peak at m/z 625 ([M-H-146]▬
). This clearly
indicated the removal of rhamnosyl moiety from hydroxyl group of more
sensitive C-7. MS3 fragmentation of 771-625 yielded ions at m/z 463 ([M-H-
146-162]▬
) and m/z 315 [M-H-146-162-162]▬
. This fragmentation pattern
was in contrast to rutinoside that showed a complete loss of the disaccharide.
Previous reports describe sophroside as a disaccharide in SB pulp. Therefore
compounds 2 and 3 were respectively identified as kaempherol-3-O-
sophroside-7-O-rhamnoside and isorhamnetin-3-O-sophroside-7-O-
rhamnoside (164,266) (Figure 3.17 & 3.18).
Results & Discussion
181
Table 3.27 HPLC-ESI-APCI profile of flavonoid glycoside in KMEF
Peak
No.
Flavonoids Characteristic mass
peaks (m/z)
Amount
(%)
[M-H]- MS
2
1 Quercetin-3-O-sophroside-7-O-
rhamnoside
771 625, 463, 301 2.4 ± 0.1
2 kampherol-3-O-sophroside-7-O-
rhamnoside.
755 609, 429, 285 1.3 ± 0.1
3 isorhamnetin-3-O-sophroside-7-
O-rhamnoside
785 639, 459, 314 3.7 ± 0.2
4 Quercetin-3-O-glucoside-7-O-
rhamnoside
609 463, 447, 301 1.1 ± 0.1
5 Isorhamnetin-3-O-glucoside-7-O-
rhamnoside
625 477, 314 1.1 ± 0.0
6 Quercetin-3-O-rutinoside 609 301 5.9 ± 0.3
7 Isorhamnetin-3-O-rutinoside 623 315 4.9 ± 0.2
8 Isorhamnetin-3-O-glucoside 477 315 2.8 ± 0.1
9 Quercetin 301 286 1.1 ± 0.1
10 Kaempherol 286 271 0.6 ± 0.0
11 Isorhamnetin 315 300 2.7 ± 0,4
Total 27.3
The amounts of identified flavonoid glycosides were quantified in
terms of their aglycones using DAD and results are presented in Table 3.27.
Quercetin-3-O-rutinoside (5.9%), isorhamnetin-3-O-rutinoside (4.9%) and
isorhamnetin-3-O-sophroside-7-O-rhamnoside (3.7%) were found as the
major flavonoid glycosides in the KMEF. Significant amounts of
isorhamnetin-3-O-glucoside (2.8%) and quercetin-3-O-sophroside-7-O-
Results & Discussion
182
rhamnoside (2.4%) were also found in the extract. The aglycones
isorhamnetin, quercetin and kaempherol respectively contributed 2.7, 1.1 and
0.6% of KMEF. The identified flavonols and their glycosides together
accounted 88% (approximately) of total flavonols (determined by aluminium
chloride method).
Figure 3.17. Structures of quercetin, kaempherol and isorhamnetin
and their glycosides in SB seeds
.
Compound R R1 R2
Quercetin OH H H
Quercetin-3-O-sophoroside-7-
O- rhamnoside
OH β-D-glucose-(1,2)-D-glucose rhamnose
Quercetin-3-O-rutinoside OH α-L-rhamnose-(1,6)-D-glucose H
Kaempherol H H H
Kaempherol-3-O-sophroside-
7-O-rhamnoside
H β-D-glucose-(1,2)-D-glucose rhamnose
Isorhamnetin OMe H H
Isorhamnetin-3-O-
sophoroside-7-O- rhamnoside
OMe β-D-glucose-(1,2)-D-glucose rhamnose
Isorhamnetin-3-O-glucoside-
7-O- rhamnoside
OMe glucose rhamnose
Isorhamnetin-3-O-rutinoside OMe α-L-rhamnose-(1,6)-D-glucose H
Isorhamnetin-3-O-glucoside OMe glucose H
O
O
R
O
OH
OH
O
R1
R2
Results & Discussion
183
Figure 3.18. HPLC-M
S/M
S total ion chromatogram of flavonoid glycosides in K
MEF
NB
: P
eak
s 1,2
,3…
. C
hec
k w
ith T
able
. 3.2
7
Results & Discussion
184
3.4.3.2 In vitro AO capacity of major flavonols in KMEF
LC MS/MS analysis showed that quercetin, kaempherol, isorhamnetin
and their glycosides were the AO active compounds in the ethyl acetate
fraction of kernel methanol extract. Therefore the AO activity of pure
quercetin, kaempherol, isorhamnetin was evaluated and results are
summarized in Table 3.28. Among the studied flavonols quercetin (IC50 = 12
nM) had the highest DPPH● scavenging efficacy. Isorhamnetin and
kaempherol respectively had IC50 values 26 and 35 nM. TEAC was also
higher for quercetin (11 nM) than isorhamnetin (7 nM) and kaempherol (5
nM). Fe(III) reducing power of the flavonols was in the decreasing order of
quercetin (1.2 nM), kaempherol (0.9 nM) and isorhamnetin (0.7 nM). Fe(II)
chelation capacity of 5 nM of flavonols was compared. Highest Fe(II)
chelation was shown by quercetin (50%) followed by isorhamnetin (39%) and
kaempherol (34%). Hydroxyl radical scavenging capacity follows the order
quercetin (IC50 = 61 nM), kaempherol (IC50 = 86 nM) and isorhamnetin (IC50
= 112 nm) IC50 values for SOS efficacy of quercetin, kaempherol and
isorhamnetin was respectively 35, 54 and 66 nM respectively. Kaempherol
(IC50 = 51 nm) had the highest XO inhibition capacity than isorhamnetin (IC50
= 59 nm) and quercetin (IC50 = 73 nm). Among the studied flavonols
quercetin showed significantly higher AO capacity than other two. DPPH*
scavenging and XO inhibition efficacies and TEAC of flavonols followed an
order of quercetin > isorhamnetin > kaempherol. An activity order of
quercetin > kaempherol > isorhamnetin was obtained for hydroxyl and SOS
and Fe(III) reduction capacities.
The ease of oxidation and AO activity of many flavonoids and their
derivatives has been demonstrated in variety of oxidation systems for many
years. Radical scavenging and metal ion complexation are suggested as
mechanisms of their AO action. Electron transfer from the flavonoid to the
Results & Discussion
185
unfilled electron deficient orbital of oxidants followed by a rapid proton
transfer is suggested as the mechanism of their AO activity (268).
The product, phenoxyl radical is stabilized by resonance delocalization
of the unpaired electrons in the ring. Repeated studies have shown that
flavonoids having greater number of hydroxyl groups or hydroxyl groups
localized ortho to one another are more effective AOs. The B ring of the
flavonoids is usually the initial target of oxidants as it is more electron rich
than A and C rings, whose electron densities are somewhat drained away by
the carbonyl group. Glycosylation of one or more of the hydroxyl groups of a
flavonoid greatly reduces its AO activity. Unsaturation in C ring (C2 – C3
bond) enhances the stability of flavonoid phenoxyl radical, hence the AO
activity. C2 – C3 double bond provides elongated conjugated double bond
system and also results in increased planarity of molecule. Both factors favor
the stability of flavonoid phenoxyl radical hence the radical scavenging (268).
The free radical scavenging by flavonoids is highly dependent on the presence
of a free hydroxyl group at C3. Lower activity of flavones with similar
hydroxyl configuration of flavonols demonstrates the effect of C3 hydroxyl
group. In addition to reduction in the number of hydroxyl groups, removal of
the C3 hydroxyl affects the conformation of molecule. Flavonols and flavanols
with C3 hydroxyl group are planar while the flavones and flavanones lacking
this feature are slightly twisted. Planarity permits conjugation, electron
dislocation and a corresponding increase in flavonoid phenoxyl radical
stability. The blocking of C3 hydroxyl group in C ring by the substitution of
3-OH by methyl and glycoside groups decreases the AO activity of flavonoids
(269).
PhOH DPPH● PhO● DPPH + +
Results & Discussion
186
In this study significant AO activity shown by quercetin, kaempherol
and isorhamnetin could be attributed to the presence of large number of
hydroxyl groups including C-3 hydroxyl group along with C2 – C3 double
bond. Quercetin, kaempherol and isorhamnetin are only differed in their B
ring substitution pattern. Presence of orthodihydroxy groups in quercetin at 3’
and 4’ positions could be the reason for its significantly high AO activity over
kaempherol and isorhamnetin. Slightly higher activity of isorhamnetin over
kaempherol in DPPH● scavenging and TEAC could be due to the presence of
methoxyl group at 3’ position. The presence of bulky methoxyl group ortho to
the hydroxyl group might favored the release of H● from hydroxyl group at 4’
carbon. Lower solubility of isorhamnetin might be the reason for its slightly
lower hydroxyl and SOS capacities and Fe(III) reduction power.
XO inhibitory activity of flavonoids depends on their hydroxyl group
configuration and planarity of molecule. Hydroxyl groups at C-5 and C-7 and
double bond between C-2 and C-3 are the important contributing factors to
flavonoid’s XO inhibition capacity. The C2 – C3 double bond keeps the
flavonoid molecule planar. The presence of hydroxyl group at C-3 position
slightly decreases the inhibition (270). Significant inhibitory activity shown
by quercetin, kaempherol and isorhamnetin in this study could be attributed to
their hydroxyl groups at C-5 and C-7 positions and C2 – C3 double bond. The
inhibitory activity of flavonols (kaempherol > isorhamnetin > quercetin) was
in confirmation with the previous results (270).
Results & Discussion
187
Table 3.28: In vitro AO capacities of flavonols (mean±SD).
3.4.3.3 Chemical Profiling of KMWF.
Tannins constitute greater proportions of high polar fractions of
phenolics and accounts for a major class of phenolics in plant kingdom.
Tannins are formed by the condensation of simple phenolics. Tannins are
generally classified into hydrolysable tannins and condensed tannins or PAs.
Hydrolysable tannins are high molecular weight derivatives of phenolic acids
mainly gallic and ellagic acid. Gallic acid and ellagic acid derivatives are
respectively called gallotannins and ellagitannins. Both gallotannins and
ellagitannins are derived from pentagalloyl glucose but the latter has
hexahydroxydiphenyl units and liberates ellagic acid on hydrolysis.
Tannins are the least studied class of phenolics in SB. Due to the high
molecular size and complexity in structure resolution and subsequent analysis
of tannins are difficult. In this study, therefore the tannin rich water fraction
of kernel was hydrolyzed and phenolic acid composition was analyzed using
Activity Quercetin Kaempherol Isorhamnetin
DPPH* scavenging IC50 (nM ) 12 ± 2 35 ± 6 26 ± 3
TEAC (nM) 11 ± 2 5 ± 1 7 ± 1
Fe(III) reducing /nM (AAE) 1.2 ± 0.1 0.9 ± 0.1 0.7 ± 0.1
% of Fe (II) Chelation/5nM 50 ± 6 34 ± 3 39 ± 2
Hydroxyl radical scavenging.
IC50 (nM) 61 ± 9 86 ± 11 112 ± 12
SOS. IC50 (nM) 35 ± 5 54 ± 6 66 ± 3
XO Inhibition IC50 (nM) 73 ± 14 51 ± 19 59 ± 22
Results & Discussion
188
HPLC. PAs in the fraction was further fractionated on sephadex gel column
chromatography and characterized in terms of prodelphinidin: procyanidin
ratio and ADP.
3.4.3.3a Phenolic acid composition of KMWF
Phenolic acid composition of KMWF hydrolysate is presented in Table
3.29. The phenolic acid profile thus obtained included the phenolic acids
liberated not only from the tannins but also from other low molecular weight
glycosides and esters. Gallic acid was the predominant (5.39%) among the
phenolic acids in KMWF. Gallic acid constituted 62.7% of total phenolic acid
in KMWF, indicating that gallotannins are the major hydrolysable tannins in
SB kernel. Low concentration of ellagic acid (0.39%) showed comparatively
lower amount of ellagitannins. Reports on the tannin profile of SB seeds are
not available hence the discussion with reference to previous report was not
attempted. Isolation and utilization of SB leaf hydrolysable tannins as an
antiviral drug under the trade name ‘Hiporamin’ have been reported (271).
Detailed studies on the biological and chemical properties of seed
hydrolysable tannins are warranted.
Phenolic acids other than gallic acid and ellagic acids were mainly
liberated from their low molecular weight glycosides and esters. The amount
of these phenolic acids in KMWF decreased in the order of protocatacheuic
acid (1.25%), vanillic acid (0.90%), p-hydroxybenzoic acid (0.56%) and
ferulic acid (0.39%). Small amounts of cinnamic acid, p-coumaric acid and
caffeic acid were also detected in the extract (Table 3.29, Figure 3.11).
Results & Discussion
189
Table 3.29: Phenolic acid composition of KMWF
Composition Amount (%)
Gallic Acid 5.39 ± 0.12
Protocatechuic Acid 1.25 ± 0.14
p-hydroxy benzoic acid 0.56 ± 0.09
Vanillic Acid 0.90 ± 0.08
Ellagic acid 0.03 ± 0.00
Cinnamic Acid 0.14 ± 0.02
P- Coumaric Acid 0.01 ± 0.00
Ferulic Acid 0.39 ± 0.05
Caffeic Acid 0.05 ± 0.01
Total 8.59
Total phenolic acid content in the extract was 8.59% accounting for
41.3% of total phenolics. This indicated that hydrolysable tannins along with
other low molecular weight phenolic acid derivatives contributed to the AO
capacity of KMWF. In vitro AO capacity evaluation of gallic acid in this
study and previous reports confirmed the AO role of phenolic acids
derivatives (268). This study also showed high DPPH●, ABTS
● scavenging
and Fe(II) reduction capacity for gallic acid. Further, moderate SOS and XO
inhibitory activities were demonstrated for gallic acid. Radical scavenging
and metal chelation are suggested as the mechanism of AO action of phenolic
acids. Number and configuration of hydroxyl groups particularly,
orthodihydroxy groups and extended conjugation in side chain (eg; cinnamic
acid derivatives) are the major contributing factors for their radical
scavenging efficacy. Among the naturally occurring phenolic acids, gallic
acid with its three hydroxyl groups is probably the most readily oxidized and
Results & Discussion
190
would be expected to the best AO (268). As a constituent of gallotannins, it
presumably contributes significantly to the AO activity of plants.
3.4.3b. Characterization of PAs in KMWF
Total PA content in KMWF was found to be 12.8%, which constituted
60% of total phenolics in the extract. Therefore characterization of PAs in the
extract was attempted. PAs are natural polymeric plant products composed of
flavon-3-ols linked together by carbon-carbon bonds. PA structure varies
considerably with unit; inter flavonoid bond location and branching.
Modification with non -flavonoid substitutes and molecular weight
distribution also contribute to this variation. Owing to this complexity and
high molecular weight complete characterization of PAs still remains a
difficult task in natural product chemistry. Several colorimetric methods have
been proposed for their quantitative analysis (272). Depolymerisation of PAs
in the presence of nucleophiles is frequently employed for the structural
analysis of PAs (273). The nucleophile forms adduct with the chain extender
units in the polymer, which are purified or analyzed by chromatography.
Their structure once established can be used to determine the nature of
monomer units within the polymer. Numerous nucleophiles such as thioacetic
acid (274), benzene-p-sulphonic acid (275), benzyl mercaptan (276),
mercaptanol (277) and phloroglucinol (278) have been used for the
characterization of PAs. Phloroglucinol is usually preferred to the sulphur
nucleophile because it is odorless (273).
Size exclusion chromatography is exclusively used for the resolution of
PAs. In this study KMWF was subjected to size exclusion chromatography
using sephadex gel. One fraction of KMWF PAs was purified by washing
with ethanol. The purified PA fraction (KPAF) was eluted with 25% acetone
(v/v) and subjected to depolymerisation in presence of a nucleophile
(phloroglucinol). The reaction products were analyzed in LC-MS/MS.
Results & Discussion
191
Purified PA of SKWF was hydrolyzed in presence of phloroglucinol
(PG) and the reaction products were analyzed in HPLC-DAD-MS/MS. Under
acidic conditions, PAs become depolymerized, releasing terminal sub units as
flavan-3-ol monomers and extension sub units as electrophilic flavan-3-ol
intermediates. The electrophilic intermediates can be trapped by nucleophilic
reagents such as phloroglucinol to generate analyzable adducts. Reaction
products were identified by analyzing the uv and mass spectra and by
comparing with the available reports (165,279). Amounts of individual
products were quantified from the calibration plots obtained with catechin and
epicatechin. ESI-MS conditions were optimized with catechin. A negative
mode ESI-MS analysis was performed (Figures 3.19 & 3.20).
Peaks 1 and 2 respectively belonged to ascorbic acid and PG (Figure
3.20). Compounds 3 and 4 resulted in a similar mass spectra with a pseudo
molecular ion peak at m/z 429 ([M-H]▬
) and ions at m/z 465 ([M+Cl] ▬
).
MS/MS fragmentation of m/z 429 produced daughter ions at m/z 303 ([M-H-
C6H6O3] ▬
) indicating lose of phloroglucinol moiety, and Retro Diels-Alder
product at m/z 261 ([M-H-C8H8O4] ▬
). Based on these mass spectra
compounds 3 and 4 were assumed to be either gallocatechin phloroglucinol or
epigallocatechin phloroglucinol. Comparing the retention data of these
compounds with available reports, compound 3 and 4 were respectively
assigned to be gallocatechin-(4α-2)-phloroglucinol and epigallocatechin-(4β-
2)-phloroglucinol adducts (Figure 3.19) (165).
Compounds 6 and 7 (Figure 3.20) also resulted in similar mass spectra.
Pseudo molecular ions at m/z 413 ([M-H]▬
) along with m/z at 449 ([M+Cl]
▬) indicated the presence of (epi) catechin phloroglucinol. MS/MS analysis of
m/z 413 resulted in daughter ions at m/z 287 ([M-H-C6H6O3] ▬
) (loss of PG)
and at m/z 261 ([M-H-C8H8O4] ▬
) (RDA fission) further confirmed the
Results & Discussion
192
assigned structure. From the retention data compounds 6 and 7 respectively
assumed to be catechin-(4α -2)-phloroglucinol and epicatechin-(4α -2)-
phloroglucinol (Figure 3.19) (279).
Compounds 5 and 8 (Figure 3.20) with molecular ion peaks at m/z 305
([M-H]▬
) and daughter ions at m/z 287 ([M-H-H2O]▬
) and m/z 179 ([M-H-
C6H6O3] ▬
) were assumed to be (epi)gallocatechin (279). Comparing with the
retention data compounds 5 and 8 were respectively assigned as gallocatechin
and epigallocatechin. Compounds 9 and 10 were identified as catechin and
epicatechin by comparing with authentic standards. Calibration curves using
catechin and epicatechin were plotted and used for the quantification of
reaction products (Figure 3.20).
Quantification of reaction products were performed using DAD
responses. Epigallocatechin-phloroglucinol (21.3mM) and gallocatechin-
phloroglucinol (20.3) adducts respectively had higher concentration among
the reaction products. 5.6 and 2.5 mM of catechin and epicatechin
phloroglucinol adducts were respectively found in hydrolyzed fractions.
Gallocatechin (1.0 mM), epigallocatechin (1.8 mM), catechin (0.2 mM) and
epicatechin (0.7 mM) were also detected in the fraction.
Results & Discussion
193
Table 3.30. Concentration of reaction products after acid catalyzed
cleavage of KMWF proanthocyanidins in presence of phloroglucinol.
Compounds Characteristic mass peaks
(m/z)
Conc.
(mM)
[M-H]▬ [M+Cl]
▬ MS
2
gallocatechin-(4α-2)-phloroglucinol 429 465 303,261 20.3
epigallocatechin-(4β-2)-phloroglucinol 429 465 303,261 21.3
catechin-(4α-2)-phloroglucinol 413 449 287,261 5.6
epicatechin-(4β-2)-phloroglucinol 413 449 287,261 2.5
Gallocatechin 305 - 287,179 1.0
Epigallocatechin 305 - 287,179 1.8
Catechin 289 - - 0.2
Epicatechin 289 - - 0.7
Prodelphinidin – procyanidin ratio 4.9 : 1
PAs can be classified into 6 sub units (procyanidins, prodelphinidins,
profisetinidins, propelargonidins, prorobinetidins and proguibourtinidins)
depending on the hydroxyl substitution pattern of single favan-3-ol units
Procyanidins consist of catechin and epicatechin extension units and
prodelphinidins of gallocatechin and epigallocatechin sub units. Acid
catalysed depolymerisation of SB seeds mainly resulted (epi)catechin and
(epi)gallocatechin derivatives, showing that mainly procyanidins and
prodelphinidins constituted seed PAs. Therefore prodelphinidin – procyanidin
ratio as ratio of (epi)gallocatechin to (epi)catechin derivatives, was estimated
from the concentrations of reaction products. The purified fraction of kernel
PA found to have a prodelphinidin – procyanidin ratio of 4.9:1 (Table 3.30).
Some reports on the composition of SB seed PA were published
recently (165,167,168). Since these reports describe the composition of
Results & Discussion
194
different fraction of seed PA, no quantitative correlation could be expected
between these reports. The present study dealt with the composition of
purified total PA in seed kernel and results are in qualitative agreement with
the available reports.
Figure 3.19. Structures of reaction products obtained by the
acid catalyzed cleavage of SB PAs in presence of phloroglucinol.
Compound R R1 R2 R3
Catechin H OH H H
Epicatechin H H OH H
Gallocatehin OH OH H H
Epigallocatechin OH H OH H
catechin-(4α -2)-phloroglucinol H OH H Ph.G
epicatechin-(4β-2)-phloroglucinol H H OH Ph.G
gallocatechin-(4α-2)-phloroglucinol OH OH H Ph.G
epigallocatechin-(4β-2)-phloroglucinol OH H OH Ph.G
HO OH
OH
O
R1
OH
OHHO
OH
R
R2
R3
Phloroglucinol (Ph.G)
Results & Discussion
195
Figure 3.20. Total ion chromatogram of depolymerisation reaction
mixture of purified PAs from KMWF
AsAc: ascorbic acid, PhG: phloroglucinol, GC-PhG: gallocatechin-
phloroglucinol, EGC-PhG: epigallocatechin-phloroglucinol, GC; :
gallocatechin, C-PhG: catechin-phloroglucinol, : EC-PhG :
epicatechin-phloroglucinol, : EGC: epigallocatechin, Cat: catechin, E
Cat: epicatechin
Results & Discussion
196
3.4.3.3c Fractionation of KMWF
Until now many chromatographic separation methods of high
molecular weight polyphenols especially PA have been developed, but there
is no perfect method satisfying separation efficiency, simplicity and scale of
treatment. Most of these methods fail in the resolution of high molecular
weight PA (280). Size exclusion chromatography is considered as a relatively
simple and efficient technique to resolve moderately high molecular weight
polyphenols. In this study resolution of KMWF using sephadex gel size
exclusion chromatography was attempted. Most of the non phenolics in
KMWF were washed out with 100% ethanol. Low molecular weight
phenolics including PAs up to trimers have been reported to be eluted with
ethanol (280). Therefore ethanol eluents were collected as fraction 1.
Increasing concentration of water in acetone was used to elute remaining high
molecular weight polyphenols mainly PA.
3.4.3.3d. Yield and composition of KMWF fractions Yield and composition of fractions are presented in Table 3.31. A
major fraction of KMWF was eluted as F-1 (48.4%). F-4, F-5 and F-3
respectively yielded 10.9, 3.1 and 1.3% of KMWF. Other fractions had < 1%
yield.
Phenolic content analysis showed that fractions except F-1 had higher
concentration of phenolics than that in KMWF. High yield of F-1 with low
phenolic concentration showed that most of the non-phenolics in KMWF
were eluted with ethanol as F-1 and most of the phenolics (> 70%) were
distributed between the remaining fractions. Fractions F-3, F-7 and F-8
together had more than 90% phenolic concentration. F-2, F-4, F-5 and F-6
found to contain 60 – 80% phenolics. F-4 accounted for the highest
proportion (32.4%) of phenolics in SKWF, followed by F-1 (28.6%), F-5
(10.1%) and F-3 (6.2%).
Results & Discussion
197
Table 3.31 Yield and composition of KMWF fractions.
Fractions Yield (%) TPC (%) PAs (%) ADP
1 48.4 12.3 ± 0.9 1.0 ± 0.3 1.96
2 0.9 80.2 ± 1.9 37.4 ± 0.9 3.09
3 1.3 98 ± 2.2 97.8 ± 4.5 3.75
4 10.9 61.9 ± 2.9 62.9 ± 1.9 3.89
5 3.1 70.8 ± 1.4 80.5 ± 1.6 5.76
6 0.8 74.1 ± 3.2 58.5 ± 0.7 5.88
7 0.2 96.7 ± 0.9 99.1 ± 5.1 7.05
8 0.3 96.1 ± 3.7 20.0 ± 0.9 8.54
PA content in these fractions was analyzed by vanillin-HCl estimation
method. Similar to phenolic concentration, all the fractions except F-1
contained higher amount of PA than that in KMWF. In contrast to phenolics
F-1 accounted only for 3.8% PA in KMWF. This indicated that phenolics
other than PA were preferentially eluted with ethanol. Ethanol fraction might
also contain low molecular weight PA. Most of the PAs in KMWF was found
as eluted with various proportions of acetone and water. F-3 and F-7 had more
than 95% PA concentration. F-5, F-4 and F-6 respectively contained 80.5,
62.9 and 58.5% PA. Fractions 3 to 5 accounted for 82% of total PAs in
KMWF. F-4 alone contributed 32.4% of PA in KMWF. Close association of
values obtained for the phenolic and PA contents in F-3, F-4, F-5 and F-7
indicated that PA constituted most of the phenolics in these fractions. Of the
96% of phenolics in F-8, PA contributed only 20% indicating that F-8
contained polar phenolics other than PA. These results showed that size
exclusion chromatographic technique used here is effective for the preparation
of phenolics especially PA enriched fractions.
Results & Discussion
198
ADP of PA fractions. The greater parts of biological and chemical properties
of PA are governed by their chemical structure and size. Therefore ADP of
the PA fractions as an indication of their molecular size were estimated as the
ratio of absorbance of the anthocyanins formed by the oxidative
depolymerisation of PA to the absorbance of vanillin PA adduct. The
fractions of water extract obtained by the sephadex gel chromatography were
evaluated for their ADP and summarized in Table 3.31. The fractions 1 to 8
showed a gradual increase in their polymerization index. ADP varies from
1.96 (F-1) to 8.54 (F-8). Fractions 3 to 5 with ADP varying from 3.75 to 5.76
accounted for 82% of total PAs in KMWF. The elution of PA in the
increasing order of ADP showed that the chromatographic mode is adsorption
rather than size exclusion.
3.4.3.3e. AO capacities of KMWF fractions
KMWF fractions obtained by the size exclusion chromatography were
subjected to AO capacity studies. DPPH● scavenging capacity evaluation
showed that all the fractions except F-1 had higher radical scavenging activity
(IC50 ≤ 3.5µg) than KMWF (IC50 = 4.1 µg). F-3, F-7 and F-8 showed higher
activity than reference gallic acid. (IC50 = 1.6 µg) (Table 3.32). DPPH●
scavenging capacity of fractions except F-8 was in correlation with their
phenolics and PA content. These results showed that PA was the major
phenolic AOs in fractions 1-7. F-8 might have some radical scavenging
phenolics other than PA. No direct correlation was observed between the
DPPH● scavenging capacity and ADP of fractions (Table 3.31 & 3.32).
TEAC values of 1 mg of KMWF fractions are summarized in Table
3.32. F-3, F-4 and F-7 showed significantly higher TEAC values (approx. 14
nM). F-1 (4.6 nM) had the lowest TEAC value. F-2, F-5 and F-6 respectively
had the TEAC values 4.7, 7.3 and 4.4 nM. In contrast to DPPH●
scavenging
activity F-8 had lower TEAC value than that of 2 to 7 fractions. TEAC
Results & Discussion
199
values of fractions did not show any dependence on the ADP of PAs in the
fractions.
Fe(III) reducing power of KMWF fractions was compared in terms of
ascorbic acid equivalents (Table. 3.32). Fractions 2 to 8 had higher reducing
power than KMWF. F-3 (8.74 µg) had the highest reducing power followed
by F-5 (5.61 µg), F-8 (5.43 µg), F-4 (5.42 µg) and F-6 (4.81 µg). Reducing
power also did not show any direct correlation with degree of polymerization
of PA fractions. Activity of most of the fractions was higher than gallic acid.
Fe(II) chelation capacity of the fractions were determined as the
stability of their complexes. The % of Fe(II) chelated by 1 mg of fractions is
presented in Table.3.32. F-2 (65 %) showed the highest chelation capacity
followed by F-3 (53 %), F-5 (48%) and F-6 (47 %). 1 mg of F-1 and F-4
respectively chelated 39.6 and 16.4% of Fe(II) in the system. No significant
Fe(II) chelation capacity was showed by F-7 and F-8 under experimental
conditions. There was no correlation between Fe(II) chelation capacity of
fractions and their ADP.
Hydroxyl radical scavenging capacity of the fractions is presented in
(Table.3.32). Fractions 2 to 6 had higher scavenging capacity than KMWF. F-
3 (17 µg) showed the highest activity and the activity of other fractions
decreased in the order of F-4, F-2, F-6 and F-8.
SOS capacity of the fractions was evaluated and given in Table.3.32.
All the fractions except F-1 showed higher SOS activity than KMWF. Among
the KMWF fractions F-3 (IC50 = 11.8 µg) had the highest activity followed by
F-2 (IC50 = 12.2 µg), F-4 (IC50 = 14.2 µg) and F-7 (IC50 = 19.8 µg). Fractions
F-5 (IC50 = 38.2 µg), F-6 (IC50 = 27.8 µg) and F-7 (IC50 = 25.4 µg) also
showed high SOS efficacy.
Results & Discussion
200
All the fractions found to inhibit the XO inhibitory activity in a dose
dependent manner. F-2 (IC50 = 3.9 µg) had higher XO inhibition capacity than
allopurinol. All the fractions showed higher inhibition activity than KMWF.
The IC50 values of remaining fractions varied between 17.0 to 71.9 µg.
Inhibition activity of the fractions did not show any dependence on the degree
of polymerization.
Sephadex gel size exclusion chromatography was effective in resolving
PAs in increasing order of molecular size. Fractions F-3 to F-5 found to
contribute larger portion of PA in the kernel. These fractions with ADP 3.7 to
5.8 had significantly high AO capacity among the fractions. No direct
correlation was observed between the ADP and in-vitro AO efficacy of
fractions. Number of phenolic groups has a positive correlation with radical
scavenging efficacies of phenolic compounds. In this case molecular size and
number of phenolic groups per molecules increased from fractions 2 to 7.
Molecular size and stereochemistry also contribute to the activity of
molecules. After certain limits solubility of high molecular weight compounds
decreases with molecular size. Steric hindrance to active sites of molecules
increases with molecular size. These opposing factors might contribute to the
poor correlation between ADP and in vitro AO capacities of PA fractions.
Results & Discussion
201
Table 3.32: In vitro AO capacities of KMW
F fractions (m
ean±SD N=5).
Fractions
DPPH
Radical
scaven
ging
capacity
IC50 (
µg
)/m
L
TEAC
(nM)
% of Fe(II)
Chelation/1
mg
Fe(III)
reducing
power/100 µ
g
[AA
E (
µg)]
Hydroxyl
radical
scavenging
capacity
IC50 (
µg
)
SOS
IC50 (
µg
)
XO
Inhibition
IC50 (
µg
)
1
10
.9 ±
0.5
4
.6
± 0
.4
39.6
± 1
.1
83
± 0
.04
1
31
± 1
2
63
.5
30
.3
2
3.5
± 0
.4
4.7
±
0.2
6
5.0
± 3
.4
831
± 0
.86
22
± 5
1
2.2
3
.9
3
1.5
± 0
.2
14
.2
± 0
.7
53.0
± 7
.1
874
± 0
.65
17
± 4
1
1.8
17
.03
4
2.9
± 0
.2
14.0
±
1.1
1
6.4
± 2
.5
542
± 0
.36
21
± 2
1
4.2
5
7.6
5
3.9
± 0
.5
7.3
±
0.9
4
8.0
± 1
.9
561
± 0
.94
37
± 4
3
8.2
4
4.0
6
2.8
± 0
.2
4.4
±
0.1
4
7.0
± 3
.4
481
± 0
.69
23
± 3
2
7.8
6
0.0
7
0.9
± 0
.9
14
.5
± 0
.3
NS
6
12
± 1
.02
61
± 4
1
9.8
48
.0
8
1.5
± 0
.7
3.0
±
0.7
N
S
543
± 0
.86
78
± 5
2
5.4
7
1.9
202
Tannins are wide spread in plant derived foods and thus may be
important dietary AOs. Polymeric polyphenols are reported as more potent
AOs than simple phenolics. Tannins are reported to have little or no pro-
oxidant effects, whereas many small phenolics are pro-oxidants (281). Several
biological effects are attributed to PAs. Most of these activities are related to
their AO activities. Faria et al; demonstrated a positive correlation between
AO activity of grape PA fractions and their capacity to inhibit cell
proliferation (282). Tannins form strong complexes with protein and are
resistant to degradation. Tannins are important AOs in digestive tract by
sparing with other AOs and thus indirectly increasing the AO levels in other
tissues. Tannins may also protect proteins, carbohydrates and lipids in
digestive tract from oxidative damage during digestion (283).
This is the first comprehensive study on the in vitro AO capacities of
SB seeds and their correlation with the chemical composition of active
extracts. Chemical finger printing of AO active fractions of SB seeds using
modern analytical techniques such as LC-MS/MS was attempted here for the
first time. Polyphenols particularly flavonols, phenolic acid derivatives and
PAs were found as the major AO compounds in SB seeds. Phenolics are
generally nontoxic and manifest a diverse range of beneficial biological
activities. The role of phenolics in cancer prevention, cardioprotection and
prevention of neurodegenerative diseases are widely discussed. Several
studies have shown that phenolics are extensively metabolized in vivo
resulting a significant alteration in their AO capacities (283). The
physiological significance of dietary AOs depends on their mechanism of
absorption and biotransformation, thus warranting further investigations on
the bioavailability of SB phenolics. Isolation of major active compounds and
subsequent activity evaluation is required in order to gather knowledge on
their structure activity relationships.
203
Chapter 4
SUMMARY AND CONCLUSION
Summary & Conclusion
204
Let food be thy medicine, thy medicine shall be thy food
..........Hippocrates
Summary & Conclusion
205
4.1. The inverse association between intake of fruits and vegetables and
delayed onset of chronic diseases are well documented by epidemiological
studies. Recently detailed studies on the chemical composition of fruits and
vegetables and its relation with their physiological activities attained much
interest among researchers. The knowledge on interactions between
phytochemicals and physiological systems are particularly important in
assessing their biological benefits as well as their possible toxic effects. In the
present study SB (H. rhamnoides) berries were investigated from this
perspective. SB, particularly in India, is a underutilized medicinal plant.
Detailed chemical profiling of H. rhamnoides berries, quality evaluation of
berries belonging to major SB species grown in India, development of a green
integrated process for processing of fresh berries for nutraceutical and
cosmaceutical applications, evaluation of AO activity of berry extracts and
chemical profiling of active fractions were therefore set as the main objectives
of the present study. The results from this investigation thus could facilitate
commercial utilization of Indian SB berries.
4.2 Detailed chemical profiling of SB berries belonging to the most common
species in India i.e. H. rhamnoides was conducted for the first time. The
berries were separated into pulp, seed coat and kernel and analyzed in detail.
They were characterized in terms of their proximate composition, lipid
profile, phenolic and organic acid profiles. Pulp and seed showed
considerable variations in terms of their proximate composition. GC analysis
of pulp and seed oil FAs indicated unique FA profiles for both oils. The
occurrence of large proportion (48.2%) of palmitoleic acid in pulp oil is
unique among the oils of plant origin. Seed oil was rich in the two essential
fatty acids, linoleic (29.5%) and α-linolenic acids (26.2%). Pulp oil was found
to have significantly higher amount of carotenoids (2548 mg/Kg) whereas
seed oil contained moderate amount of carotenoids (389 mg/Kg). HPLC
analysis showed that both pulp and seed oils were rich in tocols. Pulp oil had
Summary & Conclusion
206
1394 mg/kg of tocols with α-tocopherol accounting for 70% along with β-
tocopherol, δ-tocopherol, γ-tocopherol, α-tocotrienol, γ-tocotrienol and δ-
tocotrienol in minor amounts. Seed oil with 1193 mg/kg of tocols also had α-
tocopherol as the major tocol. In contrast to the pulp oil, seed oil had high
amount of γ-tocopherol. Presence of α-tocotrienol, β-tocopherol and δ-
tocotrienol were also detected in seed oil. HPLC analysis of sterols shown
that β-sitosterol and stigma sterol were the major sterols in pulp and seed oils.
Seed oil (1.62%) had significantly higher amount of phytosterols than that of
pulp oil (0.41%). Fresh SB Berries were highly acidic in nature with 3.6%
(malic acid equi.) titrable acids. Quinic acid isolated from the berry pulp
along with other organic acids was used to optimize RP-HPLC-DAD
analytical conditions for the profiling of organic acids. HPLC analysis
showed that quinic acid (2.80%) as the major organic acid in SB berries,
along with malic acid (1.60%), citric acid (0.16%) and traces of fumaric acid
and maleic acids. HPLC analysis also revealed that Indian berries (H.
rhamnoides) also contained high levels of vitamin C (2232 mg/kg of fresh
wt.).
4.3 Detailed profiling of polyphenols in H. rhamnoides berries were also
attempted. Pulp, Seed coat and kernel respectively contained 2.73, 3.95 and
8.58% of phenolics on dry weight. A flavonol named isorhamnetin was
isolated from the hydrosylate of kernel methanol extract. RP-HPLC-DAD
conditions for the quantitative profiling of SB flavonols were optimized. Pulp,
seed coat and kernel respectively had 288, 18 and 553 mg/100g of flavonols.
Quercetin, kaempherol and isorhamnetin were identified as the major
flavonols in the SB berries. PAs were found as a major class of phenolics in
SB berries. PAs constituted 46, 22 and 51% of total phenolics in pulp, seed
coat and kernel respectively. Extraction conditions for the preparation of PA
rich extracts were optimized. ADP of purified PA factions of pulp, seed coat
Summary & Conclusion
207
and kernel were respectively 7.4, 8.2 and 5.6. RP-HPLC-DAD conditions for
the qualitative and quantitative profiling of phenolic acids in SB berries were
optimized and validated. Phenolic acids in the berries were separated into
free, glycosides and esters and analyzed. Gallic, protocatechuic, p-
hydroxybenzoic, vanillic, salicylic, cinnamic, p-coumaric, ferulic and caffeic
acids were identified and quantified in the extracts.
4.4 The quality of SB berries belonging to major species and different geo-
climatic regions in Indian Himalayas were compared in terms of their
composition of bioactive components. SB berries belonging to H.
rhamnoides, H. tibetana and H. salcifolia species were collected from the
cold deserts of Himalayas (Lahaul, Ladakh and Spiti) and characterized in
terms of their chemical profile of pulp oil and vitamin C and phenolic content.
Composition of SB berries varied significantly from species to species. Total
carotenoids varied from 692 to 3420 mg/kg in pulp oils of fresh berries. The
amount of total tocols in the pulp oils ranged from 666 to 1788 mg/kg. H.
salicifolia berries showed small amount of pulp oil with lower levels of
carotenoids and tocopherols. No much difference in the proportion of
individual tocols in pulp was noticed among the varieties. α-tocopherol alone
constituted 40-60% of total pulp tocols in berries. Pulp oils had palmitoleic
(32 – 53%) as the most abundant fatty acid followed by palmitic (25 – 35%),
oleic (8 – 26%), linoleic (5 – 16%) and linolenic (0.6 – 2.6%), with highest
deviation observed in the proportion of palmitoleic acid in these berries. H.
rhamnoides and H. tibetana species contained the highest amount of
lipophillic constituents like carotenoids and tocols. H. salicifolia berries had
significantly higher amount of lipophobic constituents like vitamin C and
flavonols. This is the first comprehensive report on the systematic chemical
profiling of SB berries of Indian origin. For the first time the distinct nature of
H. salcifolia berries, which is found only in Himalayan region, with high
content of hydrophilic components like flavonols and vitamin C and very low
Summary & Conclusion
208
content of oil and lipophilc components like carotenoids and tocopherols was
revealed. Nutritional quality of berries of H. tibetana, with its limited
distribution in India, is reported here for the first time.
4.5. An efficient process for the production of nutraceutical and cosmaceutical
products from fresh SB berries was developed at pilot scale and process
streams and products were chemically evaluated. In this process fresh berries
were subjected to high-pressure continuous dewatering using screw press. The
separated liquid phase containing 80–90% of pulp oil was clarified at 80o
C
and centrifuged to obtain pulp oil, clear juice and sludge. The pulp oil yield
was 2.7–2.8% of fresh berry weight with 66–70% extraction efficiency. The
pulp oil was remarkably rich in carotenoids, tocopherols and sterols, with a
characteristic fresh berry flavor with palmitoleic acid as the major fatty acid.
The clear juice obtained was free from oil and contained high amounts of
vitamin C and other phytochemicals such as polyphenols and flavonoids.
Isorhamnetin along with quercetin and kaempherol were the major flavonoid
in the juice. The juice was very acidic (pH 3), with high concentrations of
organic acids. 50 to 80% of the bioactive phytochemicals in the pulp was
found to be retained with juice. Supercritical CO2 conditions for the extraction
of oil from the seeds were optimized. The seeds separated from the pressed
cake were subjected to supercritical CO2 extraction. Chemical composition of
seed oil thus obtained was compared with that of oil extracted with hexane.
Such a product development approach with out using chemicals and organic
solvents with high retention of the bioactive phytochemicals in the end
products is reported for the first time. The process was developed at pilot
scale and techno-economic feasibility was established.
Summary & Conclusion
209
4.6 In the present study it was attempted to fill the gap in the knowledge on
AO capacity of the berries and their chemical composition. AO activity
guided fractionation of berries and chemical profiling of active fractions were
attempted. The berry extracts particularly kernel extracts showed high AO
activities and high yield. Activity guided fractionation showed that major AO
active phytochemicals in kernel were concentrated in its ethyl acetate and
water fractions of methanol extract. Therefore, these fractions were further
subjected to detailed chemical profiling.
4.7. Phenolics especially flavonoids were found as the major active
components in the ethyl acetate fraction. HPLC-DAD-MS/MS conditions for
the analysis of flavonoid glycosides were therefore optimized and ethyl
acetate fraction was subjected to HPLC-MS/MS analysis. Nine flavonoid
glycosides and 3 flavonols were identified as the major components in the
ethyl acetate extract. The identified flavonoids could account for the major
fraction (approx. 88%) of phenolics in the ethyl acetate fraction. This is the
first report on the LC-MS profiling of SB seed flavonoids.
4.8. Water fraction was analyzed for its tannin composition as tannins are the
major high polar phenolics in the plants. Water fraction was hydrolyzed and
subjected to HPLC analysis for phenolic acid composition. Gallic acid
constituted 62.7% of total phenolic acids in the fraction indicating that
gallotannins could be the major hydrolysable tannins in kernel. Total PA
content in water fraction constituted 60% of total phenolics in the extract.
Therefore characterization of PA the extract was also conducted. PAs in the
water fraction was purified and subjected to depolymerisation in presence of a
nucleophile (phloroglucinol). The reaction products were analyzed in LC-
MS/MS. Epigallocatechin-phloroglucinol and gallocatechin-phloroglucinol
adducts had higher concentration among the reaction products. Catechin and
epicatechin phloroglucinol adducts and free gallocatechin, epigallocatechin,
Summary & Conclusion
210
catechin and epicatechin were also detected in the hydrolysed fraction. PAs in
the kernel fraction was found to have a prodelphinidin – procyanidin ratio of
4.9. Resolution of kernel PAs according to their molecular size was also
attempted with the help of size exclusion chromatography. The fractions thus
obtained were subjected to AO capacity assays and characterized in terms of
their degree of polymerization. PAs were found to fractionate in their
increasing order of molecular size. Most of the PA fractions had significantly
high AO activity. No direct correlation was observed between AO capacity of
these fractions and degree of polymerization.
Quest for health friendly phytochemicals as nutraceuticals, food
additives, chemopreventive and therapeutic agents is far stronger than ever
before. On the commercial point of view not many natural sources have been
investigated for their therapeutic and chemopreventive efficacy and chemical
composition. Availability and renewability of plant sources over
petrochemical based synthetic molecules enhance their importance. SB grown
in the cold deserts of Indian Himalayas with its high nutritional quality and
therapeutic efficacy, in India is an under utilized natural resource. In vitro AO
efficacy of SB berries in relation to its chemical composition was investigated
in this study. Evidence for their therapeutic and chemopreventive effects of
SB berries and other plant parts warrants further studies using in vivo models.
Detailed investigations on chemical composition particularly tannin and
protein composition are also suggested for the future work. Systematic
approach for the collection, storage, processing and value addition of SB plant
products would be helpful in the economic development of tribal areas in the
Himalayan foot hills where SB is mostly grown.
211
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List of Abbreviations
ABTS 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)
ACN Acyl carbon number
ADP Average degree of polymerization
ALA α-linolenic acid
AO Antioxidant
APCI Atmospheric pressure chemical ionization
BHA Butylated hydroxyanisole
BHT Butylated hydroxytoluene
CVD Cardio vascular disease
DAD Diode array detector
DPPH 2,2-Diphenyl-1- picrylhydrazyl
ECD Electrochemical detector
EGCG Epigallocatechin gallate
ESI Electrostatic ionization
ESR Electron spin resonance
ET Ellagitannin
FA Fatty acid
F-C Folin-Ciocalteau’s
FID Flame ionization detector
GAE Gallic acid equivalent
G-I Gastro-intestinal
GPL Glycerophospholipid
HAT Hydrogen atom transfer
HPTLC High Performance Thin Layer Chromatography
KME Kernel methanol extract
KMEF Ethyl acetate fraction of kernel methanol extract
KMWF Water fraction of kernel methanol extract
KWE Kernel water extract
LDL Low density lipoprotein
MDA Malondialdehyde
220
PA Proanthocyanidin
PDA Photo diode array
PDT Photo dynamic therapy
PG Propyl gallate
PUFA Poly unsaturated fatty acids
PME Pulp methanol extract
PWE Pulp water extract
RNS Reactive nitrogen species
ROS Reactive oxygen species
RP Reverse phase
SB Sea buckthorn
SC-CO2 Super critical carbondioxide
SCME Seed coat methanol extract
SCWE Seed coat water extract
SD Standard deviation
SET Single electron transfer
SOD Superoxide dismutase
SOS Superoxide radical scavenging
TAG Triacylglyceride
TBHQ t-butyl hydroxy quinone
TEAC Trolox equivalent antioxidant capacity
TR Retention time
XO Xanthine oxidase
221
PUBLICATIONS
Papers
1. A. Ranjith, K. Sarin Kumar, V.V. Venugopalan, C. Arumughan, R. C.
Sawhney, and Virendra Singh.
Fatty Acids, tocols and carotenoids in pulp oil of three sea buckthorn
species (H. rhamnoides, H. salcifolia and H.tibetana) grown in Indian
Himalayas.
Journal of American Oil Chemists Society,Vol 83 (2006) 359-364.
2. Ranjith Arimboor, V.V. Venugopalan, K. Sarin Kumar, C. Arumughan,
and R. C. Sawhney.
Integrated processing of fresh Indian sea buckthorn (Hippophae
rhamnoides) berries and chemical evaluation of products.
Journal of the Science of Food and Agriculture, Vol.86:2345–2353 (2006)
3. Ranjith Arimboor, K. Sarin Kumar and Arumughan C.
Simultaneous estimation of phenolic acids in sea buckthorn (Hippophae
rhamnoides) using RP-HPLC with DAD.
Journal of Pharmaceutical and Biomedical Analysis, Vol. 47 (1), 31-38
(2008).
4. Divya Sukumar, Ranjith Arimboor and Arumughan C
HPTLC finger printing and quantification of lignans as markers in sesame
oil and its polyherbal formulations.
Journal of Pharmaceutical and Biomedical Analysis, Vol. 47 (2008) 795–
801.
5. S.G. Aravind, Ranjith Arimboor, Meena Rangan, Soumya N. Madhavan
and C. Arumughan
Semi-preparative HPLC preparation and HPTLC quantification of
tetrahydroamentoflavone as marker in Semecarpus anacardium and its
polyherbal formulations
Journal of Pharmaceutical and Biomedical Analysis,
DOI:10.1016/j.jpba.2008.08.008.
222
Papers/ Posters presented in International Seminars
1. Studies on quality aspects of Indian Sea buckthorn and product/process
development. International Seminar on Sea buckthorn, a resource for
environment, health and economy, New Delhi, 2004. (Paper)
C. Arumughan, V. V. Venugopalan, A. Ranjith, K. Sarin Kumar, R. C.
Sawhney, O. P. Chaurasia and D. P. Attrey.
2. Integrated Processing of Fresh Indian Sea buckthorn (Hippophae
rhamnoides) Berries and Chemical Evaluation of Products. International
seminar on Sea Buckthorn, China, August 2005. (Paper)
C. Arumughan, V. V. Venugopalan, A. Ranjith, K. Sarin Kumar, R. C.
Sawhney, O. P. Chaurasia and D. P. Attrey.
3. HPLC-DAD-APCI-ESI-MS analysis of flavonoid glycosides in antioxidant
active extracts of sea buckthorn (H. rhamnoides) seeds.
International Symposium on Pharmaceutical and Biomedical Analysis (PBA
2009), Agra, India. March 1 – 4, 2009. (Poster)
Ranjith arimboor and C Arumughan.
........miles to go before I sleep and I have promises to keep........miles to go before I sleep and I have promises to keep........miles to go before I sleep and I have promises to keep........miles to go before I sleep and I have promises to keep....